Fruit Manufacturing

6 . Chemical Composition of Fruits and its Technical Importance 141 Calcium Apple Apricot Avocado Tangerine 40 Banana Sapote Blackberry Sapodilla Breadfruit Raspberry 30 Quince Plum 20 Carissa Pineapple 10 Cherimoya Pear Cherry,Sour Red Peach 0 Cherry, Sweet Passion -Fruit Crabapple Papaya Fig Orange Gooseberry Mulberry Grapefruit Meloncantaloupe Guava Mango Loquat Jackfruit Lime Jujub Kiwifruit Lemon Figure 6.10. Calcium content in 100 g edible portion of fruits (Watt and Merrill, 1963; Wills, 1987). Mineral substances are present as salts of organic or inorganic acids, or as complex organic combinations (chlorophyll, lecithin, etc.); they are in many cases dissolved in cellular juice. Mineral-rich fruit includes strawberries, cherries, peaches, and raspberries. Important quantities of potassium (K) and absence of sodium chloride (NaCl) give a high dietectic value to fruits and to their processed products. Phosphorus is mainly supplied by vegetables. Potassium is a major mineral present in fruits and ranges from 30 (cherimoya) to 600 mg/ 100 g (avocado) edible portion (Fig. 6.11). Phosphorus works with calcium to make strong bones and teeth. A diet that has enough protein and calcium also provides enough phos- phorus (Fig. 6.12). Fruits with relatively high levels of sodium and magnesium are shown in Figs. 6.13 and 6.14, respectively. Copper is of particular interest since it is a cofactor for PPO and can also serve as a catalyst for numerous oxidative reactions. Iron helps to build red blood cells and aids the blood in carrying oxygen to the cells. Iron content of selected fruits is given in Fig. 6.15. 6.1.6. Vitamins Many reactions in the body require several vitamins, and the lack or excess of any one can interfere with the function of another. As the body cannot manufacture all vitamins, it must absorb them from food. Vitamins are also added to fruit products mainly for nutritional purposes.

142 Fruit Manufacturing Potassium Watermelon LONGANS Tangerine 400 Loquat Strawberry Lychee 300 Mango Soursop 200 Melon honeydew Sapote Mulberry Sapodilla 100 Nectarin 0 Rose apple Orange Rhubarb Papaya Raspberry Passion fruit Quince Peach Pomegranate Pear Plum Persimmon Pineapple Figure 6.11. Potassium content of selected fruits (100g edible portion) (Watt and Merrill, 1963; Wills, 1987). High P fruits (mg/kg) Strawberry Apricot Avocado Soursop 700 Banana Sapote 600 Blackberry Passion fruit 500 Breadfruit 400 300 200 Mulberry 100 Cherimoya 0 Lychee Cherry, sweet Loquat Gooseberry Longans Guava Kiwifruit Jackfruit Jujub Figure 6.12. Selected fruits with high phosphorous content (Watt and Merrill, 1963; Wills, 1987).

6 . Chemical Composition of Fruits and its Technical Importance 143 Strawberry Sodium Mammy apple Soursop Avocado Melon cantaloupe Sapote 30 Melon honeydew Mulberry Sapodilla 20 10 0 Rhubarb Passion fruit Quince Persimmon Figure 6.13. Selected fruits with high sodium content (100g edible portion) (Watt and Merrill, 1963; Wills, 1987). Magnesium Watermelon Longans Loquat Tangerine 30 Lychee Strawberry 25 Mango 20 Soursop 15 Melon honeydew Sapote 10 Mulberry Rose apple 5 Nectarine 0 Rhubarb Orange Raspberry Papaya Quince Passion fr, purple Pomegranate Peach Plum Pear Pineapple Persimmon japan Figure 6.14. Selected fruits with high magnesium content (100g edible portion) (Watt and Merrill, 1963; Wills, 1987).

144 Fruit Manufacturing Metal concentration (mg/kg)2 Rose apple1.8 Papaya Copper Tangerine1.6 Zinc Grapefruit1.4 Iron Mango1.2 Persimmo Watermelon1 Cranberry 0.8 Rhubarb Carambola0.6 Pomegranate Gooseberry0.4 Cherry, Sour 0.2 Fig Stberry0 Kiwifruit Cherimoya Breadfruit Raspberry Lemon Soursop Sapodilla Avocado Mulberry Figure 6.15. Metal composition of selected fruits (Wills, 1987; Nagy, 1990; Somogyi et al., 1994). Vitamin B group and vitamin C (or ascorbic acid) are water-soluble vitamins that are not stored in the body for long, hence should be consumed every day. Table 6.5 lists major vegetable source of vitamins, and Fig. 6.16 shows a selection of fruits with relatively high ascorbic acid content. Some vitamins also serve multiple functional purposes: vitamins C and E act as antioxi- dants, prevent undesirable color changes, and retard the development of rancidity. Provita- min A (or b-carotene) and riboflavine (vitamin B2) are used as natural colorants. Four vitamins (A, D, E, and K) are known as the fat-soluble vitamins. They are digested and absorbed with the help of fats that are in the diet. 6.1.7. Water Water plays an active part in many chemical reactions and is needed to carry other nutrients, regulate body temperature, and help eliminate wastes (Dauthy, 1995). Water makes up about 60% of an adult’s body weight. Requirements for water are met in many ways. Most fruits are more than 80% water (Fig. 6.17). 6.1.8. Aroma Aroma components exist in very small quantities in fruits and are composed of various chemical species: alcohols, aldehydes, esters, terpenes, etc. Ripe fruits, especially bananas, Table 6.5. Major vegetable sources of vitamins. Vitamin Major vegetable sources Vitamin B1(thiamine), mg Cereal grains, nuts Vitamin B6, mg Cereal grains Folic acid, mg Green leafy vegetables, wheat bran and germ Citrus fruits, green peppers, broccoli, cantaloupe Vitamin C, mg Vegetable oils, margarine, cereal grains Green leafy vegetables, vegetable oils Vitamin E, IU Vitamin KÃ IU is international unit, mg stands for milligram, Ã indicates no data available. Source for RDA data: US Department of Health, Education, and Welfare.

6 . Chemical Composition of Fruits and its Technical Importance 145 300 250 200 150 100 50 0 Ascorbic acid (g/kg) Guava Kiwifruit Orange Lychee Lemon Lime Strawberry Mandarin Quince Grapefruit Carambola Mango Pineapple Cherry Passion Fruit Tomato Figure 6.16. Ascorbic acid content of selected fruits (Watt and Merrill, 1963; Wills, 1987). Tomato Water Apricot Strawberry Apple Avocado Quince 95 Banana Pumpkin 90 Carambola Pomegranate 85 Cherry 80 Plum 75 Fig 70 Pineapple 65 Grape 60 Persimmon Grapefruit Pear Guava Peach Jackfruit Passion Fruit Kiwifruit Orange Lemon Olive, green Mango Lime Nectarine Lychee Melon honeydew Mandarin Figure 6.17. Water content of fruits (100g edible portion) (Wills, 1987; Nagy, 1990; Somogyi et al., 1994).

146 Fruit Manufacturing Table 6.6. Esters usually found in fruits. Name Formula Identified in Butyl-acetate CH3COOC4H9 Bananas Octyl-acetate CH3COOC8H17 Oranges Ethyl-butyrate CH3COOC2H5 Pineapples oranges, and pineapples, owe their odors to the presence of esters. Some common esters formed with acetic acid (CH3COOH) are found in Table 6.6. Fruit processing techniques must be designed and operated to reduce loss and modification of aroma components. The volatile components of fruit aroma are usually recovered by removing them by a partial evaporation of the fresh juice, prior to the clarification and/or concentration oper- ations. These dilute aqueous aromas are usually concentrated by distillation and then returned to the juice. The process of aroma recovery and concentration can be optimally designed and efficiently operated if the composition of the aroma is known. Figure 6.18 compares the aroma quality of volatile extracts obtained from whole and peeled apples, based only on the most desirable compounds (Carelli and Lozano, 1989). Results indicated that, in the case of the GS aroma, only those volatiles with relatively high retention times increased their relative composition when the whole fruit was processed. On the contrary, ‘‘whole RD apple’’ aroma was rich in those desirable compounds with low retention times. However, organoleptic assessment indicated ‘‘whole apple’’ aroma to be more ‘‘fruity’’ and ‘‘characteristic’’ than that of peeled apples independent of the apple variety. Figure 6.19 compares the volatiles present in the aroma extracted from a commercial apple essence. 120 1/2 h 100 1 h Recovery (%) 80 60 40 20 0 Ethylbutyrate Ethanol Hexanol Butanol 2-Methyl-i-butanol Ethyl acetate Hexanol Benzanal Figure 6.18. Contribution of peel to aroma: comparison between desirable volatiles of ‘‘whole apple’’ and ‘‘peeled apple’’ aroma. Expressed as relative area (A%) of total desirable (Adapted from Carelli and Lozano, 1989.) A very significant increase in ethanol content was also determined and attributed to extensive fermentation during the grinding and pressing operations.

6 . Chemical Composition of Fruits and its Technical Importance 147 1000Volatile (ppm) 100 10 1 0.1 0.01 0.001 4-Methoxyallyl-benzene Acetophenone Benzanal Hexyl acetate Trans-2-hexnol Ethylisobutyrate Trans-2-hexenal Hexanol Butyl acetate 2-Methyl-1-butanol Ethyl valerate +pentyl acetate Hexanal Propanol+ethly butyrate Ethyl acetate Butanol Ethanol Figure 6.19. Volatile composition of apple aroma (Source: Carelli and Lozano, 1989). The values indicated that substantial losses of very valuable components (e.g., ethyliso- butyrate, pentyl acetate, and trans-2-hexenal) occurred during the industrial process. 6.1.9. Color compounds Different pigments complete the proximate composition of fruits. Color is an important aspect of both natural and processed fruits. Natural colorants are in general unstable, and color of fruits and fruit products may change during processing and storage. Natural pigments may be defined as the pigments occurring in unprocessed fruits, as well as those formed upon processing and storage. Major fruits and fruit products pigments can be grouped into chlorophylls, carotenoids, flavonoids (anthocyanins and anthoanthins), Melanoi- dins, and caramels (Dauthy, 1995). . Chlorophyll. This is the most abundant of all these pigments. In living plant tissues, chlorophyll is present in chloroplast cells as colloidal suspension and is associated to carbohydrates and protein. There are two types of chlorophyll: (a) Blue-green. Its chemical formula is C55H72O5N4Mg. (b) Yellow-green. Found in most green tissues; its formula is C55H70O6N4Mg. In many fruits chlorophyll is present in the unripe state and gradually disappears during ripening. Chlorophyll is water insoluble. Sodium copper chlorophyllin salt, obtained after the hydrolysis of chlorophyll with sodium hydroxide and the replace- ment of magnesium with copper, is a heat-stable coloring food. . Carotenoids. Pigments belonging to this group are fat soluble and range in color from yellow through orange to red. Important fruit carotenoids include the orange carotenes

148 Fruit Manufacturing of apricot, peach, and citrus fruits; the red lycopene of watermelon and apricot; and the yellow-orange xanthophyll of peach. These and other carotenoids seldom occur singly within plant cells. The content of these pigments rarely exceeds 0.1%. In fruits, b-carotene is an indication of provitamin A content. The carotenoids g, b-carotene, and phytofluene were reported as the three main carotenoids in passion fruit (purple) (Chan, 1994). . Flavonoids. Flavonoids are polyphenolic compounds possessing 15 carbon atoms; two benzene rings joined by a linear three-carbon chain. This class of pigments are water soluble and commonly present in the juices of fruits. Flavonoids include the purple, blue, and red anthocyanins of grapes, berries, plump, eggplants, and cherries; the yellow anthoxanthins of apples, and the colorless catechins and leucoanthocyanins, which are tannins present in apples and grapes. These colorless tannin compounds are easily converted to brown pigments upon reaction with metal ions. Anthocyanin pigments (red and purple) occur in the sap of cells. Anthocyanins give the familiar color to fruits such as red apples, blueberries, cherries, cranberries, strawberries, and plums. Anthocyanins are responsible for color in most berries. Anthocyanin concen- tration is usually expressed as cyanidin-3-glucoside/100 g pulp sample. The color of concord grapes is due to anthocyanin pigments, the major contributor being delphidin monoglucoside (McLelland and Acree, 1992). Phenolic acids are not practically found in free forms in plants because the carboxyl groups are very active and easily transform into esters or amides when combined with aliphatic alcohols and phenols or amino compounds. Phenolic acids are divided into two subgroups, which are the derivatives of hydroxybenzoic acid and hydroxycinnamic acid. The most important derivatives of hydroxybenzoic acid are ferulic acid, caffeic acid, and coumaric acid, which occur in trace amounts naturally in plants. The caffeic acid ester of d-quinic acid is one of the most important polyphenols naturally occurring in apples. Flavonoids are divided into five subgroups according to their chemical structure (Fig. 6.20). All flavonoids are derived from flavan (2-phenol-benzo-dihydropyran). The general structure (C6ÀC3ÀC6) is given in Fig. 6.21. Anthocyanidins Anthocyanidins are found in nature as glycosides and are called anthocyanins. Anthocya- nins are natural color pigments, which have a color range varying from rich red to blue, the characteristic color of most fruits. Flavones and Flavonols Flavones and flavonols, which have slight yellow color, are practically found in all plants. Flavones differ from flavonols in the absence of OH-group at C3-atom of the center ring. Flavonones Flavonones do not have a double bond at the center ring. Their glycosides are mainly found in citrus fruits, like naringin (Fig. 6.22).

6 . Chemical Composition of Fruits and its Technical Importance 149 Anthocyanidins Found in nature as Familiar Color of red glycosides called apples, blueberries, anthocyanins cherries, cranberries, strawberries, and plums Flavones and Flavones and flavonols Flavones differ from flavanols (slight yellow in color— practically found in flavonols in the absence any plants) of OH-group at C3-atom of the center ring Flavanones Flavonones do not Their glycosides are Flavonoids Catechins and have a double bond at mainly found in citrus leucoanthocyanidins the center ring fruits The most frequent catechins are dia- stereoisomer pair (+)– catechin and (–)–epicatechin, as well as (+)-gallocatechin and (–)–pigallocatechin Proanthocyanidins Catechins are very They can condense reactive if exposed to chemically and atmospheric oxygen enzymatically into oligomers and polymers, forming proanthocyanidins Figure 6.20. General classification of flavonoids (Source: Herrmann, 1976; Cook and Samman, 1996). 8 1 23 7 o 21 6 4 5 36 5 4 o Figure 6.21. General structural formula of flavonoids. Aa-L/ Rh(1-6)-b-Gl-O O OH OH O Figure 6.22. Chemical structure of naringine. Catechins and Leucoanthocyanidins Catechins are flavan-3-ol monomers comprising one OH-group at C3-atom. The leu- coanthocyanidins flavan-3,4-diols, have one OH-group at C3- and C4-atoms. The most frequent catechins are diastereoisomer pair (þ)-catechin and (À)-epicatechin, as well as (þ)- gallocatechin and (À)-epigallocatechin. Catechins are very reactive if exposed to atmospheric

150 Fruit Manufacturing oxygen. They can condense chemically and enzymatically into oligomers and polymers forming proanthocyanidins. Caramel. It is an amorphous dark brown-coloring material formed by heating sacchar- ides, alone or in the presence of amino products or selected accelerators. Maillard-type reactions are involved and the products are extremely complex in composition. Detailed information on nonenzymatic browning is given in Chapter 7. Finally, proanthocyanidins are colorless if their chain is short. Yellowish to brown color is formed with increasing polymerization. When they are heated in acidic media, they transform into corresponding anthocyanidins, getting the typical reddish-violet color. 6.2. INFLUENCE OF PROCESSING AND STORAGE ON THE COMPOSITION OF FRUITS As soon as fruits are harvested, deterioration of quality attributes or nutrients begins and increases with time. Nutrients are lost by: (a) Food processing operations (b) Sensitivity of nutrients to pH, oxygen, light, and heat (c) Enzyme action (d) Measures to control enzyme activity, e.g., blanching. 6.2.1. Vitamin Destruction During Processing and Storage Vitamin losses may occur in canned fruits when stored at high temperatures (>378C). Vitamin C (ascorbic acid) is probably the most unstable vitamin, and it is readily oxidized by many nonenzymatic processes. Although frozen storage temperatures between À18 and À288C result in satisfactory vitamin C retention levels in fruits, at temperatures above À108C, it is easily oxidized and will be drastically reduced in a short period of time. The use of package materials impermeable to oxygen and light is recommended. The enzyme ascorbate oxidase, which is not present to any great extent in vitamin C sources, causes oxidation. It was also found that the mixing of orange juice with mashed bananas naturally containing phenolase, act similar to ascorbate oxidase. All vitamins are subject to enzyme hydrolysis and the above illustrates the point. Particular food combinations can lead to nutrient loss. Benterud (1977) studied thermal stability of vitamins in a hot melt of carbohydrates free of oxygen. The author found some vitamins were extremely heat resistant (e.g., vitamin E, riboflavine), whereas thiamine was the most labile of the vitamins. Some vitamins (A, D, B12, and C) show a gradual degradation as temperature is raised from 100 to 1308C. However, in fruit processing vitamin stability condition is more complicated because pH, oxygen, ions, reducing agents, etc. influence the rate of decomposition. Figure 6.23 shows the loss in canned fruit products. Canned or bottled fruit juice stored at ambient temperature for extended period is likely to lose all its vitamin C content (Cameron, 1978). The principal causes for vitamin C destruction in bottled juice are oxidation by residual air in the head space, anaerobic decomposition, and the effect of light. It was observed that after some fruit processing operation significant loss of ascorbic acid occurs (Nagy, 1980). Up to 47% loss in vitamin C occurred in canned fruits after two years’ storage at 278C. Adisa (1986) studied the influence of storage and molds on the ascorbic acid content of orange and pineapple fruits. He found that about 40% loss of ascorbic acid was recorded in both fruits stored at 308C for 8 weeks. The rate of loss of vitamin C was observed to be faster in fruits infected with mold than in healthy fruits.

6 . Chemical Composition of Fruits and its Technical Importance 151 120Vitamin,%retained Vit.C 100 B6 Panth.acid 80 60 40 20 0 Furit juices Grapefurit juice Orange juice Peaches Apricots Ascorbic acid (%)Figure 6.23. Loss of vitamins C and B6, and panthotenic acid in canned fruit and fruit juices (adapted from Benterud, 1977). Maeda and Mussa (1986) indicated that ascorbic acid depletion with time in bottled and canned orange juice was almost linear. The ascorbic acid levels in canned orange juice stored for 8 weeks at room temperature were significantly higher than in bottled (glass) juice. Ascorbic acid is also present in relatively high concentration in raspberry. Ochoa et al. (1999) measured the percentage of residual ascorbic acid during long-term storage of rasp- berry pulp at 4, 20, and 378C (Fig. 6.24). These values are in general agreement with those 100 10 1 37؇C 20؇C 4؇C 0.1 Storage time(days) Figure 6.24. Semilogarithmic plot of the ascorbic acid reduction during storage of raspberry pulp at 48, 208 and 378C. Reprinted from Lebensm. Wilss. u Technol. 32(3): 149–153. Ochoa, M.R. Kesseler, A.G., Vullioud, M.B. and Lozano, J.E. Physical and chemical characteristics of raspberry pulp: storage effect on composition and color. (copyright) 1999, with permission from Elsevier. 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56

152 Fruit Manufacturing reported for other fruits and vegetables in the same range of temperatures (Villota and Hawkes, 1992). Ochoa et al. (1999) found that ascorbic acid decrease in raspberry pulp obeys the following linear regression equation: AA(%) ¼ 100eÀkt (6:1) where AA is the ascorbic acid content (%), k is the fitted reaction rate constant, and t is the storage time (days). Parameters and correlation coefficients of Eq. (6.1) are k37C ¼ 0:0980 (r2 ¼ 0:978), k20C ¼ 0:0424 (r2 ¼ 0:979), and k4C ¼ 0:0275 (r2 ¼ 0:995). Calculated values of the rate constant, k, were temperature dependent and the effect of this variable was calculated according to the Arrhenius model. The calculated activation energy for ascorbic acid degradation in raspberry pulp was Ea ¼ 6:45 kcal=mol (r2 ¼ 0:970). 6.2.2. Effect of Storage on Metal Content Acid liquid foods, as many fruit juices are, interact with the components of the container. In the case of canned juices, corrosion of tinplate increases the heavy metal content, especially tin, lead, and iron. Table 6.7 lists the increase in heavy metal content in canned orange juice. Packing of fruit juice in tin cans causes a higher contamination with heavy metals than that in paperboard boxes or laminated pouches. 6.2.2.1. Influence of Storage on Fruit Juice Aroma The effect of storage condition on fruit juice aroma has been extensively studied. Velez et al. (1993) studied changes of orange juice aroma due to storage time and temperature, by gas chromatographic analysis of nearly 40 volatile constituents. The authors found that at 08C changes in orange volatile were very slow and difficult to detect. Contrarily, at 358C the quality of the orange aroma deteriorates very fast. Figure 6.25 shows orange aroma deteri- oration of the principal volatile, at 208C after 3 months’ storage. During aroma stripping of the single-strength juice when the amount of evaporated water is in the 10 –12% range, up to 90% of the aroma compound evaporates. Moreover, only a slightly greater evaporation of volatile may be achieved by doubling the amount of water evaporated. Ve´lez et al. (1993) also found that only 7 out of 38 volatile were signifi- cantly affected by storage time and temperature. Table 6.7. Heavy metal content of orange juice when affected by type containers. Metal Fresh fruit Tin can Other containers Tin – 55 0.4 Lead – 0.16 0.1 Iron 0.1 2.75 0.16 Zinc 0.07 1.05 0.12 Copper 0.045 0.09 0.03 Adapted from Mesallam, 1987.

6 . Chemical Composition of Fruits and its Technical Importance 153 Acetoin Acetoin Terpinolene Linalool Volatile (mg/L) Alfa-pinene Ethyl butyrate Octanal Terpinene-4-ol Initial 3 Dodecanal 1 month Citronellon 2 months 0 3 months 12 Figure 6.25. Changes in the main orange volatile after 3 months’ storage at 208C (adapted from Kirchner and Miller, 1957; Petersen et al., 1998). 6.2.3. Fruit Juice Change in Amino Acid Content During Storage During storage fruit juices are exposed to temperatures that have an adverse influence on quality. In these fruit juices the major constituents believed to be involved in browning are the reducing sugars, amino acids, polyphenols, and organic acids (Joslyn, 1956; Cornwell and Wrolstad, 1981). Wolfrom, Kashimura, and Norton (1974) studied browning mixtures con- stituted by different amino acids and glucose in 1:1 molar ratio simulating orange juice stored at 658C and reported that g-aminobutiric acid and l-arginine were the main contributors to browning. del Castillo et al. (1998) found a loss of 65.1% of original amino acids’ content, when storing dehydrated orange juice (aw ¼ 0:44) at 508C for 14 days. About 78% of the loss was attributed to the amino acids in major proportions, namely proline, arginine, asparagine, and g-aminobutiric acid. Babsky et al. (1986) evaluated changes in the composition of clarified apple juice concentrate during prolonged storage at 378C. Results showed that storage caused an 87% loss in the total free amino acids, which was mostly due to decreases in glutamic acid, asparagine, and aspartic acid (Fig. 6.26). The major constituents were asparagine (Asn), aspartic acid (Asp), and glutamic acid (Glu). The other individual amino acids amounted to less than 10%. These values are similar to those found by Burroughs (1957) and Czapski (1975). Bielig and Hofsommer (1982), working with about 90 samples of apples, apple juices, and concentrates, found that every apple juice has a characteristic amino acid spectrum and no mean value can be specified. The concentration changes of total amino acids during storage of apple juice were very large (Fig. 6.27). Asp and Glu decreased more markedly. Several studies (Warmbier et al.,

154 Fruit ManufacturingAmino acids (mg/L) AA (mg/L) 500 450 Total % Retention 400 350 300 250 200 150 100 50 0 0 50 100 150 Time (days) Figure 6.26. Free amino acid composition of apple juice concentrate (758Brix). Variation during storage at 378C (adapted from Babsky et al., 1986). 350 Asn 300 Glu 250 Asp 200 150 100 50 0 0 20 40 60 80 100 120 Time (days) Figure 6.27. Decreases in glutamic acid, asparagine, and aspartic acid (adapted from Babsky et al., 1986). 1976; Spark, 1969; Eichner and Karel, 1972; Reyes et al., 1982) reported Maillard browning of reducing sugars with only one or two amino acids other than Asp, Asn, and Glu. Wolfrom et al. (1974) and Ashoor and Zent (1984) studied the influence of different amino acids in model systems. None of these studies have shown Glu and Asn to be very high browning producing compounds.

6 . Chemical Composition of Fruits and its Technical Importance 155 Buedo et al. (2001) studied the change of free amino acid (AA) composition of peach juice concentrate (PJC), as a result of the Maillard reactions. The authors observed that total AA content decreased 8, 35, and 60%, after 112 days of storage at 15, 30, and 378C, respectively (Fig. 6.28). The main constituent, asparagine, contributed 71% of the total loss while aspartic acid increased its concentration, probably as a result of the asparagine degradation. Buedo et al. (2001) also found that during storage of peach juice at 378C glutamine concentration drops 60 fold, while alanine reduces only to half at the same time and conditions. However, it must be taken into account that the contribution of each AA to the juice browning is not directly related to the consumption rate, as each AA can produce different chromo- phores, having different light absorbance characteristics (Labuza and Baisier, 1992). 6.2.4. Effect of Storage on Fruit Sugars Sucrose in an acid media, as many fruit products are, can hydrolyze under a rate correspond- ing to a first-order process (Babsky et al., 1986). The reducing sugars increased at a rate determined by the inversion of sucrose. It is well known that the rate of hydrolysis is a function of the concentration of reactants, temperature, and acid–catalyst concentration (Glasstone, 1946). If excess water is present the rate of disappearance of sucrose can be represented by a pseudo first-order reaction rate equation: S ¼ S0 exp (ÀKt) (6:2) where S0 is the initial sucrose concentration, moles/100 g concentrate, S is the sucrose concentration at time t; K is the rate constant (0:00822 dayÀ1 under studied conditions), t is the time, min. Hydrolysis, also called inversion because it is accompanied by an inversion of the angle of polarization, yields two simple sugars, d-glucose and d-fructose. The rate of appearance representing total reducing sugars is described by Eq. (5.3): R ¼ 2So(1 À eÀKt) þ Ro (6:3) where: R ¼ reducing sugars (glucose þ fructose) concentration at time t moles/100 g concen- trate, Ro is the reducing sugar concentration at t ¼ t0; and t is time, mm. AA (mg/L) after 11 days, storage 6000 Peach juice: 12؇Brix 5000 4000 3000 2000 1000 0 37؇C 30؇C 15؇C Initial Figure 6.28. Decrease in PJC’s total AA content during storage at 37, 30, and 158C (adapted from Buedo et al., 2001).

156 Fruit Manufacturing Schoebel et al. (1969) obtained experimental data on the dependence of the first-order reaction rate on pH. Figure 6.29 shows the development of sucrose and total reducing sugars during storage of concentrated apple juice, which increased in concentration in accordance with the predicted kinetics (Eq. 6.3). Hence, hydrolysis appeared to be the major cause of sucrose reduction (and reducing sugars increase) in apple juice at a rate determined by pH and temperature. Akhavan and Wrolstad (1980) verified that slight losses (6%) in total sugars occur after 112 days of storage at 378C of pear concentrate. Stadtman (1948) considered the possibility that relatively small chemical changes are required to produce brown pigment of intense color. If this is the case, the changes in reducing sugars necessary to produce large changes in color might be hard to be detectable. Beveridge and Harrison (1984) detected no loss of reducing sugar after heating 72.50 Brix-pear juice at temperatures up to 808C for 2 h. Reyes et al. (1982) found that glucose undergoes more browning than fructose with glycine at 608C and pH 3.5. Any detectable variation in the fructose/glucose ratio may indicate unbalanced consumption of these redu- cing sugars due to nonenzymatic browning reaction. 0,4 0,35 0,3 0,25 Sugar (mol/100 g) 0,2 Reducing 0,15 sugars Sucrose 0,1 0,05 0 0 20 40 60 80 100 120 Time (days) Figure 6.29. Sucrose hydrolysis and increase in reducing sugars in apple juice as a function of time of storage, at 378C (from Babsky et al., 1986 with permission).

6 . Chemical Composition of Fruits and its Technical Importance 157 6.2.5. Effect of Processing and Storage on Fruit Pigments Changes in fruits and fruit products color are extensively considered in Chapter 7. However, some aspects on fruit pigment degradation are revised here. Pigments may oxidize resulting in color fading of highly colored canned fruits. As carotenoids are highly sensitive to oxygen and light, particularly in the presence of metals such as iron, copper, and manganese, processing and storage can produce carotenoid degradation. Anthocyanins show low stability in prod- ucts manufactured from fruits. Moreover, the stability of these natural pigments is poor in dehydrated fruits, unless packaged in inert gas. Temperature, light, and initial composition of fruits are considered as responsible factors for the instability of fruit anthocyanins. For many apple varieties, red skin color is important for marketability. Apple harvest is largely based on the amount of red color consistent with the natural tendency of the variety. Unfortunately, this may not be the best time to harvest for optimum quality after storage. Whereas color development of fruit maturing on the tree generally increases with time, the fruit also begins to ripen and will not store as well. Different apple varieties showed different storage temperature optima for red color development. Precooling the tissue for 48 h at 28C (to simulate cold nights) increased the amount of red pigment that accumulated. 6.2.6. Changes in Organic Acid Content The role of organic acids appears to be essentially catalytic (Reynolds, 1965). Reduction of organic acids in apple juice was only 9% (Babsky et al., 1986). The slight decrease in acidity might be partly due to copolymerization of organic acids with products of the browning reactions. Lewis et al. (1949) also suggested that organic acids can react with reducing sugars to produce brown pigments. Sample pH did not change during storage, keeping its initial value of 3.72 + 0.02 almost constant. Urbicain et al. observed that the titratable acidity in peach juice rose with time and temperature. Major organic acids present in stone-free peaches are malic, citric, and quinic (Wang et al., 1993). The basic amino groups disappear during Maillard reaction, hence pH lowers as the reaction proceeds in systems with no buffers (Spark, 1969). In the case of peach juice, the organic acids present in major proportion act as a strong buffer, hence no variations of pH may be expected. Moreover, the consumption of AA would increase titrable acidity. Spark (1969) has reported that buffers increase browning rate, which boosts the color damage in concentrated peach juice. 6.2.7. Changes in Phenolic Compounds Phenolic compounds present in fruit products may react to form brown polymeric com- pounds (Abers and Wrolstad, 1979). If this reaction plays any role in the color development of apple juice, total phenolic content will not increase during storage as Babsky et al. (1986) found, using the Folin–Ciocalteau reagent (Singleton and Rossi, 1965). Cornwell and Wrolstad (1981) proposed that reductone compounds present in the juices interfere with the Folin–Ciocalteau reagent increasing the apparent phenolic contents. Market demands for ‘‘natural’’ juices and pulps devoid of food additives have prompted food scientists to study the quality deterioration of fruits during processing and storage. The raspberry (Rubus ideaus) is a bush fruit of the rosaceous family, whose economic importance is increasing because of the use of raspberry products in the food industry.

% Antocyanin158 Fruit Manufacturing Major problems confronted in the production of raspberry pulp include operations that may affect their properties. The color of raspberry fruits is not significantly affected by freezing and cold storage. However, when crushed, most fruit berries yield a highly pectinous pulp, releasing little free run juice with poor color stability on storage. Anthocyanins, which are responsible for color in most berries, easily degrade following various reaction mechan- isms affected by oxygen, ascorbic acid, pH, and temperature among other variables (Abers and Wrolstad, 1979; Skrede, 1985). Ochoa et al. (1999) found that total anthocyanin (TA) pigment in raspberries decreased significantly through storage, at a rate strongly dependent on temperature. After 40 days, pulp stored at 378C had lost the majority of the anthocyanins. Semilogarithmic plots of percentage of residual anthocyanin during long-term storage of raspberry pulps were linear (Fig. 6.30), showing that decrease followed first-order reaction kinetics, in accordance with previous findings in other berries (Skrede, 1985). It was suggested that anthocyanins may be destroyed either through direct oxidation by quinones formed from catechin by PPO action or through copolymerization of anthocyanins into brown pigments (tannins) formed from catechin–quinone polymerization (Jackman et al., 1987). Pigment instability is an undesirable consequence of processing of canned syrup straw- berries and products that contain them (Garc´ıa-Viguera et al., 1999). Processing was found to cause a 50% decrease in the flavanol concentration and the formation of a polar compound. The conversion of leucoanthocyanidin to anthocyanin when heated at acidic condition (Lee and Wicker, 1991) was responsible for the pink discoloration in canned fruits like lychee. A number of researchers have shown that the rate of ascorbic acid oxidation influences total anthocyanin loss in strawberry products. Loss of natural color was reported to be affected by AA content by Skalsky and Sistrunk (1973). However, studies on the effect of ascorbic acid on the destruction of anthocyanin pigment were in general carried out at elevated temperatures and under relatively low storage temperatures. No significant correl- ations were found between ascorbic acid content and any of the other quality factors. 100 10 37 ЊC 20ЊC 4ЊC 1 0 10 20 30 40 50 60 Storage time (days) Figure 6.30. Percentage of retention of anthocyanin in heritage raspberry pulp at 4, 20, and 378C during storage (from Ochoa et al., 1999). Reprinted from Lebensm. Wilss. u Technol. 32(3): 149–153. Ochoa, M.R. Kesseler, A.G., Vullioud, M.B. and Lozano, J.E. Physical and chemical characteristics of raspberry pulp: storage effect on composition and color. (copyright) 1999, with permission from Elsevier.

6 . Chemical Composition of Fruits and its Technical Importance 159 REFERENCES Abers, J.E. and Wrolstad, R.E. (1979). Causative factors of color deterioration in strawberry preserves during processing and storage. J. Food Sci. 44: 75–78. Adisa, V.A. (1986). The influence of molds and some storage factors on the ascorbic acid content of orange and pineapple fruits. Food Chem. 22: 139–146. Akhavan, I. and Wrolstad, R.E. (1980). Variation of sugars and acids during ripening of pears and in the production and storage of pear concentrate. J. Food Sci. 45: 499–506. Anonymous. (1992). Hot topic: food guide pyramid replaces the basic 4 circle. Food Technology, 46(7): 64 –67. Ashoor, S.H. and Zent, J.B. (1984). Maillard browning of common amino acids and sugars. J. Food Sci. 49: 1206– 1211. Babsky, N., Toribio, J.L. and Lozano, J.E. (1986). Influence of storage on the composition of clarified apple juice concentrate. J. Food Sci. 51: 564 –567. Benterud, A. (1977). Vitamin losses during thermal processing. In Physical, Chemical and Biological Changes in Foods Caused by Thermal Processing, Hoyem, T. and Kvale, O. (eds.). Applied Science Publishers Ltd, Essex, UK, pp. 185–201. Beveridge, T. and Harrison, J.E. (1984). Nonenzymatic browning in pear juice concentrate at elevated temperatures. J. Food Sci. 49: 1335–1339. Bielig, H.J. and Hofsommer, H.J. (1982). On the importance of the amino acid spectra in apple juices. Flussiges Obst. 2: 50–56. Buedo, A.P., Elustondo, M.P. and Urbicain, M.J. (2001). Amino acid loss in peach juice concentrate during storage. Innov. Food Sci. Emerg. Technol. 1: 281–288. Buglione, M.B. (2005). Chemical Changes During Grape Juice Processing and Storage. Doctoral Thesis. Universidad Nacional del Sur, Bah´ıa Blanca, Argentina. Burroughs, L.F. (1957). The amino-acids of apple juices and ciders. J. Sci. Food Agric. 3: 122. Cameron, D.J. (1978). Variation on storage of ascorbic acid levels in prepared infant feeds. Food Chem. 3(2): 103–110. Carelli, A.A. and Lozano, J.L. (1989). Apple aroma from Argentina: quality evaluation by capillary gas chromatog- raphy. HRC CC 12: 488– 490. Carr´ın, M.E. Ceci, L. and Lozano, J.E. (2004). Characterization of starch in apples and its degradation with amylases. Food Chem. 62: 215–223. Chan, H.T. (1994). Passion fruit, papaya and guava juices. In Fruit Juice Processing Technology, Nagy, S., Chen, C.S. and Shaw, P.E. (eds.). Agscience, Inc., Auburndale, FL, USA, pp. 378– 435. Chen, S.C. (1992). Physicochemical principles for the concentration and freezing of fruit juices. In Fruit Processing Technology, Nagy, S., Chen, C.S., and Shaw, P.E., Editors. Agscience, Inc., Auburndale, Florida. 23–25. Cook, N.C., and Samman, S. (1996). Flavonoids – chemistry, metabolism, cardioprotective effects, and dietary services. J. Nutr. Biochem. 7: 66–76. Cornwell, C.J. and Wrolstad, R.E. (1981). Causes of browning in pear juice concentrate during storage. J. Food Sci. 46: 515–519. Czapski, J. (1975). Wplw wolnych aminokwasdw na zmiany jakdsci zageszcconych sokdw joblkowych podczas prcechownwania. Prezem. Ferm. Ilolny 9: 19–26. Dauthy, M.E. (1995). Fruit and vegetable processing. Fao Agricultural Services Bulletin. 119 Food and Agriculture Organization of the United Nations, Rome. In: http://www.fao.org/documents del Castillo, M.D., Corzo, N., Polo, M.C., Pueyo, E. and Olano, A. (1998). Changes in amino acid composition of dehydrated orange juice during accelerated nonenzymic browning. J. Agric. Food Chem. 46: 277–280. Dennis, C. (ed.) (1983). Post-harvest Pathology of Fruit and Vegetables. Academic Press, San Diego, CA. Eichner, K. and Karel, M. (1972). The influence of water content on the amino browning reaction in model systems under various conditions. J Agr. Food Chem. 20: 218–223. Friend, J. (ed.) (1982). Recent advances in Biochemistry of Fruits and Vegetables. Academic Press, San Diego. Garc´ıa-Viguera, C., Zafrilla, P., Arte´s, F., Romero, F., Abella´n, P., Toma´s-Barbera´n, F.A. (1999). Colour and anthocyanin stability of red raspberry jam. J. Sci. Food. Agric. 78(4): 565–573. Glasstone, S. (1946). Textbook of Physical Chemistry. D. Van Nostrand, Princeton, NJ. Goodenough, P.W. and Atkin, R.K. (eds.) (1981). Quality in Stored and Processed Vegetables and Fruit. Academic Press, New York, NY, 398 pp. Herrmann, K. (1976). Flavonols and flavones in food plants: a review. J Food Technol. 11: 433–448. Hui, Y.H. (1991). Data Sourcebook for Food Scientists and Technologists. VCH Publisher, Inc., New York, pp. 331–410. Jackman, R.L. Yada, R.Y., Tung, M. and Speers, R.A. (1987). Anthocyanins as food colorants: a review. J. Food Biochem. 11: 201–247.

160 Fruit Manufacturing Jackson, J.M. and Shinn, B.M. (1979). Fundamentals of Food Canning Technology. AVI Publishing Company, Westport, CT. Joslyn, M.A. (1956). Role of amino acids in the browning of orange juice. Adv. Food Res. 22: 1–9. Kirchner, J.G., Miller, J.M. (1957). Canning and storage effects, volatile water-soluble and oil constituents of valencia orange juice. J.Agric. Food Chem. 5: 283–288. Konja, G., and Lovric, T. (1993). Berry Fruit Juices. In Fruit Juice Processing Technology, Ed. By Seven Nagy, Chin Shu Chen and Philip E. Shaw, Agscience, Inc. Auburndale, Florida. Labuza, T.P. and Baisier, W.M. (1992). The kinetics of nonenzymatic browning. In Physical chemistry of foods, Schwartzberg, H.G. and Hartel, R.W. (eds.). Marcel Dekker, New York, pp. 595–647. Lee, H. and Wicker, L. (1991). Anthocyanin pigments in the skin of lychee fruit. J. Food. Sci. 56: 466–468. Lewis, V.M., Esselen, W.B. and Fellers, C.R. (1949). Nonenzymatic browning of foodstuffs. Nitrogen free carboxylic acids in the browning reaction. Ind. Eng. Chem. 41: 2591–2599. Maeda, E. and Mussa, D. (1986). The stability of vitamin C (l-ascorbic acid) in bottled and canned orange juice. Food Chem. 22: 51–58. Mesallam, A.S. (1987). Heavy metal content of canned orange juice as determined by direct current plasma atomic emission spectrophotometry (DCPAES). Food Chem. 26(1): 47–58. Mc Lellan, R. and Acree, T.E. (1992). Grape juice. In Fruit Juice Processing Technology, Nagy, S., Chen, C.S. and Shaw, P.E. (eds.). Agscience, Inc., Auburndale, FL, USA, pp. 318–333. Nagy, S. (1980). Vitamin C contents of citrus fruit and their products: a review. J. Agric. Food Cherm. 28(1): 8–18. Nagy, S., Shaw, P.E. and Wardowski, W.F. (1990). Fruits of Tropical and Subtropical Origin. Composition, Properties and Uses. FSS, Florida Science source, Inc., Lake Alfred, FL, USA. Nagy, S., Chen, C.S. and Shaw, P.E. (1992). Fruit Processing Technology. Nagy, S., Chen, C.S. and Shaw, P.E., (eds.) Agscience, Inc., Auburndale, Florida. Oakenfull, D.G. (1991). The chemistry of high-methoxyl pectins. In The Chemistry and Technology of Pectin. R.H. Walter Ed. Academic Press Inc., San Diego, CA. 87–108. Ochoa, M.R. Kesseler, A.G., Vullioud, M.B. and Lozano, J.E. (1999). Physical and chemical characteristics of raspberry pulp: storage effect on composition and color. Lebensm. Wiss. u Technol. 32(3): 149–153. Petersen, M.A. Tønder, D. and Poll, L. (1998). Comparison of normal and accelerated storage of commercial orange juice. Changes in flavour and content of volatile compounds. Food Quality Pref. 9(1–2): 43–51. Reed, G. (1975). Enzymes in Food Processing, 2nd ed. Academic Press, London. Reyes, F.G.R., Poocharoen, B. and Wrolstad, R.E. (1982). Maillard browning reaction of sugar-glycine model systems: changes in sugar concentration, color and appearance. J. Food Sci. 47: 1376–1380. Reynolds, T.H. (1965). Chemistry of nonenzymatic browning II. Adv. Food Res. 14: 167–171. Salunkhe, D.K., Bolin, H.R. and Reddy, N.R. (1991). Storage, Processing, and Nutritional Quality of Fruits and Vegetables, 2nd ed., Vol. 1: Fresh Fruits and Vegetables (323 p.) and Vol. 2: Processed Fruits and Vegetables (195 pp.). CRC Press, Boca Raton, FL. Sanchez-Castillo, C.P., Dewey, P.J.S., Lara, J.J., Henderson, D.L., de Lourdes Solano and W. James, W.P. (2000). The Starch and sugar content of some mexican cereals, cereal products, pulses, snack foods, fruits and vegetables. J. Food Comp. Analysis, 13: 157–170. Schoebel, T., Tannenbaum, S.R. and Labuza, T.P. (1969). Reaction at limited water concentration. 1. Sucrose hydrolysis. J. Food Sci. 34: 324 –329. Singleton, V.I. and Rossi, J.A. (1965). Colorimetry of total phenolics with phosphomolybdic–phosphotungstic acid reagents. Am. I. Enol. Vitcul. 16: 144 –151. Skalsky, C. and Sistrunk, W.A. (1973). Factors influencing color degradation in concord grape juice. J. Food Sci. 38: 1060–1066. Skrede, G. (1985). Color quality of blackcurrant syrups during storage evaluated by Hunter L0, a0, b0 values. J. Food Sci. 50: 514–525. Somogy, L.P., Ramaswamy, H.S. and Hui, Y.H. (1996). Processing Fruits: Science and Technology, Vol. 2, Major Processed Products. Technomics Publishing Company, Inc., Lancaster, PA, USA. Spark, A.A. (1969). Role of amino acids in nonenzymic browning. J. Sci. Food Agric. 20: 308–312. Stadtman, E.R. (1948). Nonenzymatic browning in fruit products. Adv. Food Res. 1: 325–331. Swi-Bea Wu, J., Ming-jen Sheu and Tzuu-tar Fang (1992). Oriental fruit juices: carambola, Japanese apricot (Mei), lychee. In Fruit Juice Processing Technology, Nagy, S., Chen, C. S. and Shaw, P.E. (eds.). Agscience, Inc., Auburndale, FL, USA. USDA (1992). U. S. Development of Agriculture, Human Nutrition Information Service. The Food Guide Pyramid. Home and Garden Bulletin No. 252, Washington, D.C.: Government Printing Office, August.

6 . Chemical Composition of Fruits and its Technical Importance 161 USDA (2005). My pyramid. U.S. Department of Agriculture (USDA) and U.S. Department of Health and Human Services (HHS), January. http://www.nal.usda.gov/fnic/Fpyr/pyramid.html Velez, C., Costell, E., Orlando, L., Nadal, M.I., Sendra, J.M. and Izquierdo, L. (1993). Multidimensional scaling as method to correlate sensory and instrumental data of orange juices aromas. J. Sci. Food Agric. 61: 41– 46. Villota, R. and Hawkes, J.G. (1992). Reaction kinetics in food systems. In Handbook of Food Engineering, Heldman, D.R. and Lund, D.B. (eds.). Marcel Dekker, Inc., New York, pp. 65 –72. Wang, T., Gonzalez, A.R., Gbur, E.E. and Aselage, J.M. (1993). Organic acid changes during ripening of processing peaches. J. Food Sci. 58, 631–632. Warmbier, H.C., Schnickels, R.A. and Labuza, T.P. (1976. Nonenzymatic browning kinetics in an intermediate moisture model system. Effect of glucose to lysine ratio. J. Food Sci. 41: 981–985. Watt, B.K. and Merrill, A.L. (1963). Composition of Foods: Raw; Processed; Prepared. Agriculture Handbook No. 8. Consumer and Food Economics Research Division, Agricultural Research Service, USDA, Washington, DC. Wills, R.B.H. (1987). Composition of Australian fresh fruits and vegetables. Food Technol. Australia 39(11): 523–530. Wills, R.B.H., McGlasson, W.B., Graham, D., Lee, T.H. and Hall, E.G. (1989). Post harvest-An Introduction to the Physiology and Handling of Fruits and Vegetables. AVI Book, Van Nostrand Reinhold, New York. Wolfrom, M.L., Kashimura, N. and Horton, D. (1974). Factors affecting the Maillard browning reaction between sugars and amino acids. Studies on the nonenzymatic browning of dehydrated orange juice. J. Agr. Food Chem. 22: 796 –800.

CHAPTER 7 FRUIT PRODUCTS, DETERIORATION BY BROWNING 7.1. INTRODUCTION Food processing, defined by the predictability of the product–process interactions, is turning into a science. Processing and storage of fruit products affect composition in many ways. How to develop the best process knowledge of the reaction and a description of the engineering processes involved, the latter already studied in previous chapters, is required. Knowledge of deterioration factors, including the rates of deterioration, means that it is possible to find ways of lowering or stopping those deteriorative actions, thereby gaining fruit preservation. In order to maintain their nutritional value and organoleptic properties and because of technical–economical considerations, not all the identified methods against deteri- oration actually have practical applications. 7.1.1. Different Mechanisms of Deterioration Appearance, which is significantly impacted by color, is one of the first attributes used by consumers in evaluating food quality. Color may be influenced by naturally occurring pigments such as chlorophylls, carotenoids, and anthocyanins in fruits; or by pigments resulting from browning reactions. Browning of fruits and fruit products is one of the major problems in the fruit industry and is believed to be probably the first cause of quality loss during postharvest handling, processing, and storage. Browning can also adversely affect flavor and nutritional value. Extraction of fruit juices, or elaborating fruit pulps and pure´es, requires the grinding of fruit, which results in the rupturing of the fruit cells and the mixing of the fruit components with the atmospheric oxygen. The same is valid for the cutting of fruits before dehydration. This incorporation of oxygen into the fruit pulp, or on the surface of the cut fruit, causes the oxidation of the phenolic compounds to quinones, which are naturally present in the fruit tissue. The endogenous enzyme polyphenoloxidase (PPO) catalyzes this oxidation, known as enzymatic browning (EB). EB is one of the most devastating reactions for many exotic fruits, in particular tropical and subtropical varieties. It is estimated that over 50% loss in fruits occurs as a result of EB (Whitaker and Lee, 1995). Projected increases in fruit markets will not occur if EB is not understood and controlled. On the other hand, browning reactions of nitrogen compounds, mainly free amino acids and proteins, with carbohydrates cause deterioration by color and off-flavor development during processing and storage of fruits. These deteriorative reactions are generally known as nonenzymatic browning (NEB) reactions. However, browning reactions in fruits are more 163

164 Fruit Manufacturing complex than suggested by the simple classification as enzymatic or nonenzymatic, because of the large number of secondary reactions that may occur. This is reflected in the range of color produced even in the same product (e.g., raspberries may develop red or brown discolora- tions; Ochoa et al., 1999). Browning may occur in some fruits in which endogenous ascorbic acid (AA) is oxidized to dehydroascorbic acid (DHAA), which then reacts with free amino acids to yield deep brown colors by the Maillard reaction (Kacem et al., 1987). These deteriorative reactions need to be described in some more detail. Much research has been concentrated in recent years to find effective and economical ways to prevent browning in various fruit products. Concerted efforts have been made to understand the basic biochemistry involved in enzymatic browning reactions and to find practical techniques to prevent the browning reactions in fresh and processed products. On the other hand, the complexity of EB and NEB reactions, and the various compounds involved (Hodge, 1953; Spark, 1969) makes it difficult to study the reactions by means of a simple analytical chemical method. However, kinetic approach can be used in solving the problem. 7.2. ENZYMATIC BROWNING EB occurs in fruits after bruising, cutting, or during storage, and its control during the processing of fruits is of great importance to fruit manufacturing. EB is a significant problem in apples, pears, bananas, peaches, and grapes, particularly. Acceptability of browning also depends on the product: . In clarified fruit juice, like apple juice, a little browning is accepted and the typical amber-like hue is commercially desirable. . However, both apple pure´e and cloudy juice must retain the yellowish or greenish color, which characterizes the fresh product. It must be remembered that enzymatic problem is not always a problem to be avoided: the color of products such as raisins and prunes is obtained thanks to a controlled PPO reaction (Va´mos-Vigya´zo´ , 1981). 7.2.1. Phenolic Compounds and Oxidases POP is an example of an enzyme that can lower the quality of a food product by catalyzing the oxidation of phenolic compounds. The susceptibility to browning may depend on PPO activity and/or phenolic content (Coseteng and Lee, 1987). Polyphenol oxidase catalyzes the initial step in the polymerization of phenolics to produce quinones, which undergo further polymerization to insoluble dark brown polymers known as melanins. These melanins form barriers and have antimicrobial properties, which prevent the spread of infection or bruising in plant tissues. The formation of yellow and brown pigments in fruit products during EB reactions is controlled by the levels of phenols, the amount of PPO activity, and the presence of oxygen (Spanos and Wrolstad, 1992). The phenolic composition of apple, pear, and white grape juices was reviewed by Spanos and Wrolstad (1992) (see also Chapter 6). These authors classified the phenolic constituents of importance in fruit juices into two groups: (a) phenolic acids and related compounds, and (b) flavonoids.

7 . Fruit Products, Deterioration by Browning 165 During the browning of fruit tissue, the enzyme PPO, also called orthodiphenol oxidase or catecholase, catalyzes the oxidation of phenolic compounds related to catechol and contain- ing two o-dihydroxy groups to the corresponding o-quinone (Joslyn and Ponting, 1951; Vamos-Vigyazo, 1981). Relatively few of the phenolic compounds in fruits serve as substrates for polyphenol oxidase (Table 7.1). Compounds with minor differences may or may not be substrates for polyphenol oxidase. For example, Shannon and Pratt (1967) found that when comparing quercetin and dihydroquercetin, differing only in the bonding between carbons at the 2 and 3 positions, only the latter was a substrate for apple polyphenol oxidase. It was assumed that quercetin is more stable than dihydroquercetin, due to the presence of a double bond conjugated to an aromatic ring, affecting compound solubility. Most raw fruits contain polyphenols and PPOs, located in different compartments in the cell structure. When through damaging or processing (e.g., milling) enzyme, substrates and oxygen come into contact with each other, and a lot of reactions start that finally lead to the formation of insoluble brown pigments (melanins). The EB of fruit and vegetables is always considered as a quality loss of both fresh and processed food products. Simple representation of EB reactions is given in Fig. 7.1. 7.2.2. Kinetics of Enzymatic Browning PPO activity, as for most of enzymes, may be minimized by reducing agents, heat inactiva- tion, lowering the pH of the fruit product, and the presence of enzyme inhibitors, among other techniques, which are reviewed in Chapter 8. To effectively inhibit or control the EB in fruit products, an accurate determination of the kinetics of these catalyzed-oxidative reactions is required. The kinetics of deterioration can be followed through color measurements, which is a simple and effective way for studying the phenomenon. The substrate specificity of polyphenol oxidase varies in accordance with the source of the enzyme. Phenolic compounds and polyphenol oxidase are in general directly responsible for EB reactions in damaged fruits, during postharvest handling and processing. The relationship of Table 7.1. Phenolic substrates of PPO in fruits. Fruit Phenolic substrates Apple Chlorogenic acid (flesh), catechol, catechin (peel), caffeic acid, 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxy benzoic acid, p-cresol, 4-methyl catechol, leucocyanidin, p-coumaric acid, flavonol Apricot glycosides Avocado Isochlorogenic acid, caffeic acid, 4-methyl catechol, chlorogenic acid, catechin, epicatechin, pyrogallol, Banana catechol, flavonols, p-coumaric acid derivatives Eggplant Grape 4-methyl catechol, dopamine, pyrogallol, catechol, chlorogenic acid, caffeic acid, DOPA 3,4-dihydroxyphenylethylamine (dopamine), leucodelphinidin, leucocyanidin Mango Chlorogenic acid, caffeic acid, coumaric acid, cinnamic acid derivatives Catechin, chlorogenic acid, catechol, caffeic acid, DOPA, tannins, flavonols, protocatechuic acid, Peach Pear resorcinol, hydroquinone, phenol Plum Dopamine-HCl, 4-methyl catechol, caffeic acid, catechol, catechin, chlorogenic acid, tyrosine, DOPA, p-cresol Chlorogenic acid, pyrogallol, 4-methyl catechol, catechol, caffeic acid, gallic acid, catechin, dopamine Chlorogenic acid, catechol, catechin, caffeic acid, DOPA, 3,4-dihydroxy benzoic acid, p-cresol Chlorogenic acid, catechin, caffeic acid, catechol, DOPA Adapted from Marshall, Kim and Wei, 2000.

166 Fruit Manufacturing HO O OH O O HO O OH OH HO O OH OH PPO OH OH OH OH O OH PPO. O2 OH OH HO OH o-dihydroxyphenol o-quinone OH Melanins Figure 7.1. Simplified mechanism for the transformation of a diphenol to dark colored melanins by PPO. the rate of browning to phenolic content and polyphenol oxidase activity, has been reported for various fruits such as apples (Coseteng and Lee, 1987), grapes (Lee and Jaworski, 1988), and peaches (Lee et al., 1990). In addition to serving as polyphenol oxidase substrates, phenolic compounds act as inhibitors of polyphenol oxidases (Walker, 1995). Their inhibitory action decreased in the following order: cinnamic acid > p-coumaric acid > ferulic acid > benzoic acid. Although relatively few of the phenolic compounds in fruits serve as substrates for polyphenol oxidase, as catechins, cinnamic acid esters, 3,4-dihydroxy phenylalanine (DOPA), and tyrosine (Table 7.1), the stecheometry of complex reactions like EB in fruits as substrate is practically unknown. Therefore, instead of determination of consumption of reactives (phenols), or formation of products (melanins), the kinetics of color development is commonly used for studying the browning reactions. As found by Sapers and Douglas (1987), tristimulus reflectance values were strongly nonlinear, and changes in the rate of browning may be better understood when plotting colorimetric parameters against log time. EB in Golden Delicious apple juice was monitored by measuring CIE LÃ value (Lozano et al., 1994). The authors observed a significant influence of degree of ripeness on the rate of EB. Pulp made with unripe (GA) apples browned at a faster rate. This behavior was attributable to differences in AA content and PPO activity in young fruits. Figure 7.2 70 T = 5˚C 65 OA 60 MA 55 GA 50 CIE L* 45 40 35 10 100 30 Time, min 1 Figure 7.2. Relationship of CIE ¼ l value in Golden Delicious apple pulp to time and degree of ripeness at 58C. GA stands for green apple, MA for mature apple, and OV for overmature apple (Lozano et al., 1994 with permission).

7 . Fruit Products, Deterioration by Browning 167 shows the variation of CIE Là parameter as a function of log time with degree of ripeness of processed apple as a parameter, at constant temperature. The rate of luminosity decrease can be divided into three periods: (i) the first period, characterized as an induction or flat period, attributable to the inhibition action of the naturally occurring ascorbic acid (Ponting and Joslyn, 1948), (ii) the second period, which looks linear when represented in this semilog plot, attributed to the consumption of the enzymes’ substrates (Sapers and Douglas, 1987), and (iii) the third period that approaches a plateau at a time depending on the degree of ripeness of apples. It was also observed (Fig. 7.2) that the lower induction time corresponded to unripe fruits. Koch and Bretthauer (1956) also found considerable seasonal variations in the amount of AA and DHAA in apples. Another relevant information given by Koch and Bretthauer (1956) is that PPO activity is greatest in young fruits than in fully ripe apples. Lozano et al. (1994) found that a reduction in bà values and change in sign (from À to þ) in aà parameter clearly indicated that browning development occurred in apple pulp. Negative aà values were given by the green pigmentation of apples and it was pronounced in green samples. 7.2.2.1. Effect of the Temperature in the Color Change Color development during pulping of fruits certainly includes Michaelis–Menten type reac- tion kinetics followed by several reactions, both reversible and irreversible, up to the forma- tion of dark brown pigments. The combined effect of these browning reactions may result in a nonlinear behavior strongly dependent on temperature. Experimental data obtained with apples on the dependence of the rate of CIELAB Là with temperature were fitted by Lozano et al. (1994) to the equation: Là ¼ a À k log t ð7:1Þ where a and k are fitting parameters and t is the time in min. The selected range is in agreement with the second linear period in the Là versus log t plot. However, it must be noted that browning mechanism can be strongly nonlinear and simplified kinetics equations may not be applicable. Kinetic measurements on complex systems, such as fruit pulp, usually give reaction constant values, which may or may not be the dissociation constant, but it is frequently the combination of the rate constants for several steps. In general, for production of light-colored apple pulp, the time between milling of the fruit and the heat treatment must be as short as possible. 7.3. NONENZYMATIC BROWNING NEB via Maillard-type reactions is the most important route of color deterioration in fruit juices. The reaction is followed by undesirable color, odor, and flavor changes (Pribella and Betusova, 1978; Toribio and Lozano, 1984; Cornwell and Wrolstad, 1981). Three basic NEB reactions have been identified (Fig. 7.3): . Pyrolysis: which results in a burnt and inedible flavor; . Caramelization: when the simpler sugars lose water molecules from their structure, through a 1:2 and 2:3-enolization. This process is affected by pH. Through many

168 Fruit Manufacturing Pyrolysis Involves the total loss of water from the sugar molecule and the breaking of carbon–carbon linkages NEB Caramelization Maillard Heat-induced transformation of Browning reactions involve simple reducing sugars alone in a sugars, and amino acids and concentrated solution simple peptides Figure 7.3. Basic NEB reactions. intermediates, and in the pH 2–7 range, d-fructose for example can give rise to furans, isomaltol, and maltol, well-known bread crust flavor/aromas; . Maillard-type reaction: of amino acids and proteins with carbohydrates, which is discussed extensively in the following section. 7.3.1. Maillard Reactions The reaction begin to occur at lower temperatures and at higher dilutions than caramelization, as in clarified fruit juices (Toribio and Lozano, 1984). The rate can increase by 2–3 times for each 108C rise in temperature. Maillard reactions have three basic phases (Fennema, 1986): . The initial reaction is the condensation of an amino acid with a simple sugar, which loses a molecule of water to form N-substituted aldosylamine. This is unstable and undergoes the Amadori rearrangement to form 1-amino- 1-deoxy-2-ketoses (or ketosamines), which can cause complex subsequent dehydration, fission, and poly- merization reactions. One of the Maillard paths is a simple caramel reaction, catalyzed by amino acids. . The ketosamine products of the Amadori rearrangement can then react three ways in the second phase. One is simply further dehydration into reductones and dehydror- eductones, which are essentially caramel products. Second is the production of short- chain hydrolytic fission products such as diacetyl, acetol, pyruvaldehyde, etc. These then undergo Strecker degradation with amino acids to aldehydes and by condensation to aldols. Negative aromas like 2 and 3-methyl-butanal are also formed. . Third path is the Schiff’s base/furfural path. This involves the loss of 3 water mol- ecules, then a reaction with amino acids and water. These also undergo aldol conden- sation and polymerize further into true melanoidins. . These products react further with amino acids in the third phase to form the brown pigments and flavor active compounds collectively called melanoidins. These can be off-flavors. The outcome will depend not only on which amino acids and sugars are available, but also on pH, temperature, and concentration.

7 . Fruit Products, Deterioration by Browning 169 In general, high levels of amino acids favor both caramel and Maillard reactions, but dilution eliminates caramel reactions. At temperatures >1008C pyrazines are produced. High levels of polyphenols favor Strecker degradation. Table 7.2 lists principal reactions and characteristics of identified NEB reactions. Fruit juice concentrates containing more than 65% total solids are normally stable from the standpoint of fermentation at any temperature, but when stored at relatively high temperatures, NEB reactions occur. NEB via Maillard-type reactions is the most important route of color deterioration in apple juice (Czapski, 1975; Toribio and Lozano, 1984). The reaction takes place between amino acids and reducing sugars present in the juice, decreasing the alpha-amino nitrogen content followed by undesirable color, odor, and flavor changes (Pribella and Betusova, 1978; Toribio and Lozano, 1984). The same behavior was found in pear juice concentrate (Cornwell and Wrolstad, 1981), citrus juices (Kanner et al., 1982; Cornwell and Wrolstad, 1981), and intermediate moisture foods (Resnik and Chirife, 1979; Waletzko and Labuza, 1976). Color deterioration was reported for many fruit products, such as citrus juices (Rey- nolds, 1965; Kanner et al., 1982; Cornwell and Wrolstad, 1981), intermediate moisture foods (Waletzko and Labuza, 1976; Johnson et al., 1969; Czapski, 1975), and apple juice (Toribio and Lozano, 1984, 1986). Babsky et al. (1986) studied the effect of storage on the composition of clarified apple juice concentrate and concluded that natural juices were very complex mixtures in which the role of the many components in browning reactions was difficult to elucidate. Kinetics of NEB in fruits and fruit products can be simplified as seen in the following scheme (Fig. 7.4). Table 7.2. Basic Maillard-type reactions (adapted from Fennema, 1985). Stage Principal reactions Characteristics Initial (Colorless) Condensation, enolization, Reducing power in alkaline solution Amadori rearrangement. With increases. Storage of colorless 1:1 Intermediate (Strong absorption in proteins, glucose and free amino glucose–protein product near-ultraviolet range) groups combine in 1:1 ratio produces browning and insolubility Final (Red-brown and Sugar dehydration to dark-brown color) 3-deoxyglucosone and its -3, Addition of sulfite decolorizes, 4-ene, HMF, and reducing power in acidic solution 2-(hydroxyacetyl)furan; sugar develops, pH decreases, sugars fragmentation; formation of disappear faster than amino alpha-dicarbonyl compounds, acids. Positive test for reductones, and pigments amino sugars (Amadori compounds) Aldol condensations, polymerization, Strecker Acidity, caramel-like and roasted degradation of alpha amino acids aromas develop, colloidal and to aldehydes and N-heterocyclics insoluble melanoidins form, at elevated temperatures. Carbon fluorescence, reductone reducing dioxide evolves power in acid solution, addition of sulfite does not decolorize

170 Fruit Manufacturing Reactives Intermediates Final products Hexoses CO2 Amino-acids Amadori Melanoidines Organic acids compounds Glucids Aldehydes Vitamins Anthocyanins, etc. Standard Most of them very Large Absorbance on methods of visible range determination reactives and unstable are available (Exceptions: CO2 and 5-HMF) Figure 7.4. Simplified scheme for NEB reactions in most fruit juices. 7.3.1.1. Tristimulus Parameters and Absorbance as a Measurement of Browning in Fruit Juices The absorption at one fixed wavelength, although reliable for kinetics studies, is not adequate for comparing the visual color changes in both browned apple juice and model systems. For this reason, the Hunter a, b, L color parameters and C.I.E. x, y, z parameters are also measured (see also Chapter 5). In general during NEB of fruit juices, except for the differences given by the initial color, the tristimulus values are grouped within a narrow band drawn from the standard light source ‘‘C’’ so as to approach asymptotically the spectrum red locus (Fig. 7.5). This behavior could denote that the same NEB reactions occurred in juices and model systems, resulting in polymers (melanoidines) with the same color attributes. 7.3.1.2. Kinetics of Nonenzymatic Browning (NEB) The kinetics of NEB is generally dependent upon product characteristics and storage condi- tions, including: . Influence of temperature, soluble solids’ concentration, pH, acidity, and water activity. . Amino acids and reducing sugars’ content. . 5-HMF formation during NEB . Effect of polyphenols, galacturonic acid, and other minor compounds. As pointed out by Labuza and Riboh (1982) most of the quality-related reaction rates are either zero- or first-order reactions, and the statistical difference between both types may be small. Besides real fruit products, the technique of using simplified model food systems to simulate the effect of storage and processing on quality has been widely used. 7.3.1.3. Effect of Soluble Solids It is well known that by increasing the concentration of food solids (or reducing water content) browning reactions are significantly enhanced (Eichner and Karel, 1972). The

7 . Fruit Products, Deterioration by Browning 171 0.9 515 0.8 520 505 530 0.7 Green 500 545 0.6 555 495 565 0.5 Yellow 575 Y 490 0.4 Pink 590 Daylight 605 485 0.3 Red 480 780 0.2 Blue 0.1 470 0 380 0 0.2 0.4 0.6 0.8 X Figure 7.5. Effect of browning during storage of apple juice (adapted from Toribio and Lozano, 1987). occurrence of a maximum reaction rate at a certain water activity was also described (Labuza et al., 1970). A further increase in solids’ content resulted in rate decrease. It was suggested that at these high concentrations, the rate of reaction was controlled by the mobility of the reactants. Toribio et al. (1994) found that clarified apple juice has a slower nonenzymatic browning reaction rate (NEBr) at low water activities increasing up to the maximum point between aw % 0:53---0:55 (about 828Brix) as shown in Fig. 7.6. A further increase in aw significantly reduces the color formation. It is assumed, in this case, that the increase in aw tends to dilute the concentration of reactants, decreasing chemical reaction rate. This NEBr maximum is typical in nonenzymatic Maillard-type reactions. The aw values at maximum NEBr for model mixtures was found to be aw ¼ 0:87 (Labuza et al., 1970). 7.3.1.4. Effect of Reducing to Total Sugars’ Ratio (R/T) The rate of sucrose hydrolysis is a function of the concentration of reactants, temperature, and acid–catalyst concentration. However, if excess water is present as in fruit juices, the rate of disappearance of sucrose follows a pseudo-first-order reaction rate equation. As expected, an increase in the reducing sugars (fructose þ glucose) content, when keeping the total sugars’ content constant, resulted in a faster browning of model mixtures (Lozano, 1991). 7.3.1.5. Effect of the Fructose to Glucose Ratio (F/G) Lozano (1991) found that glucose was more reactive than fructose, at least during the zero- order reaction rate period in model solutions simulating apple juice. Wolfrom et al. (1974)

172 Fruit Manufacturing 0.025 37؇C 0.02 NEBr (Abs 420nm/day) 0.015 0.01 0.005 Storage time 0.5 0.6 0.7 0.8 0 days 0 40 days 0 80 days 0.1 0.2 0.3 0.4 aw Figure 7.6. NEBr as a function of water activity (aw) and time of storage (adapted from Toribio et al., 1984). working with a simulated orange juice, found that fructose had higher initial rate of browning than glucose during the initial stage of reaction but was dependent on the kind of amino acid. 7.3.1.6. Effect of Amino Acids (AA) Lozano (1991) also found that an increase of total amino acids’ content (AA) from 4.34 to 6.51 g/L, resulted in a noticeable increase in the browning rate. It can be calculated that for 1.5 times greater AA content, there was an approximately 1.5 increase of the reaction constant K. Similar results were also found during storage (Babsky et al., 1986) and processing (Toribio and Lozano, 1987) of clarified apple juice. Asparagine was found to represent nearly 70% of the total amino compounds in clarified apple juice, and 90% of it disappeared after 100 days of storage at 378C (Babsky et al., 1986). Calculated increase in reaction constant K was 5% when the Asn content was increased from 60 to 70%. Del Castillo et al., (1998) found a loss of 65.1% of original AA content, when storing dehydrated orange juice (aw ¼ 0:44) at 508C for 14 days. About 78% of that loss was attributed to the AA in major proportions, namely proline, arginine, asparagine, and g-aminobutiric acid. Buedo et al. (2001) studied the change of free amino acids’ (AA) composition of peach juice concentrate (PJC), as a result of the Maillard reactions, in particular the effect of the storage at 15, 30, and 378C for 6 weeks, since those are conditions likely to be found in commercial practice. A decrease in total AA content was observed to be 8, 35, and 60%, after 112 days of storage at those temperatures, respectively. The main constituent, asparagine, contributed to 71% of the total loss, while aspartic acid increased its concentration, probably as a result of the asparagine degradation. Decrease in total AA content was exponential, pH remained constant during the storage, while titratable acidity increased with both time and temperature, assumed by disappearance of amino groups.

7 . Fruit Products, Deterioration by Browning 173 7.3.1.7. Effect of the Content of Organic Acids The malic acid participation in the NEB, via Maillard reaction, was judged to be essentially catalytic (Reynolds, 1965). However, Lozano (1991) found that: (i) an increase in malic acid actually accelerated the rate of browning during storage, and (ii) the pH was reduced only by 0.1 unit when malic acid content was increased from 2 to 6 g/L. It must be noted that NEB reaction is not very sensitive to low pH changes in the pH ¼ 2– 4 range (O’Beirne, 1986). Titratable acidity in peach juice was observed to rise with time and temperature. Also, at 30 and 378C the increase rate is maximum on the initial storage days. Major organic acids present in stone-free peaches are malic, citric, and quinic (Wang et al., 1993). 7.3.1.8. Effect of Other Minor Components NEBr increased with ascorbic acid, because of the participation of vitamin C in the Maillard reactions. During the enzymatic clarification process of fruit juices, the natural pectic sub- stances (mainly polymers of the galacturonic acid) are broken by specific enzymes (pecti- nases), which are able to hydrolyze pectin to their basic units. This treatment is also applied during the pressing stage, to improve the juice extraction, and depending on fruit variety and maturity, considerable amount of free galacturonic acid could be present in clarified fruit juices after enzyme treatments. Lozano (1991) found that the adding of 60 mg/L of galacturonic acid to a model sugar– malic acid–amino acid solution accelerated the color formation. It can be concluded that galacturonic acid, produced during the enzymatic treatment of pulps and juices, may accel- erate browning, thus reducing the storage capacity of apple juice concentrate. 7.3.1.9. Effect of Temperature Figure 7.7 shows the change in absorbance at 420 nm for different apple juice concentrates and a model solution over 120 days, at 378C. The rate of NEB of this model solution can be divided into the following two stages: Absorbance (420 nm) 2 Red Del. Granny Smith Model system 1.5 1 0.5 37؇C 0 0 25 50 75 100 125 Storage time (days) Figure 7.7. Color development as a function of time of apple juice (708Brix) and a model solution (fructose/glucose: 3.13, reducing/total sugars: 0.90, total amino acids: 3.5 g/L, malic acid: 6.4 g/L at 128Brix) (adapted from Toribio and Lozano, 1984; Lozano, 1991).

174 Fruit Manufacturing (1) An induction period, already observed in other model food systems (Warmbier et al., 1976), is attributed to the formation of colorless intermediates. This period was exponential rather than linear and the color development could be expressed as a typical first-order reaction equation. (2) A linear period of reaction where the color formation follows a zero-order kinetics. It was assumed that the behavior with temperature would follow the same trend at any given time when the storage temperature was reached. Moreover, when concentrate was stored for 30 days at 378C and the next 30 days at 208C, color formation was 40% higher than that during the inverse condition (first 30 days at 208C and the next 30 days at 378C). Clearly this shows how important it is to cool the product as soon as it is produced. The information is relevant because a sizeable amount of concentrate is made during the summer season and the product is stored in the open air. When the color formation during storage of model solutions at any temperature is compared with the color development of a natural apple juice, some differences are readily observed (see Fig. 7.7): (1) Color increase was very much higher in AJC than in model solutions, (2) Induction time was not detected during the AJC storage, (3) Obtained maximum NEBr had different values. The first two differences could be attributable both to the influence of minor components like galacturonic acid and to the heating during processing (clarification, aroma recovery, and concentration stages), which could reduce or eliminate the induction time. Eichner (1975) and Eichner and Karel (1972) found in very viscous food systems that the viscosity remarkably affected the NEBr. As the viscosity of both the model systems and apple juice had practically the same values at the same soluble solids, it is difficult to exclusively attribute the limitation of NEB reactions and the occurrence of a maximum to the reactant mobility. It must be noticed that the use of liquid model systems for kinetic studies, besides the previously considered limitations, resulted in a very good tool to individually quantify the participation of juice components in deteriorative reactions. To put it succinctly, browning rates in fruit juice are mostly dependent on reducing sugars and amino acids’ content. However, independent of juice composition, the lower the storage temperature, the less is the darkening. Thus, from a practical standpoint, thermal history is crucial in obtaining lighter color products. 7.3.2. 5-HMF Formation During Storage and Processing of Fruit Products The Maillard reaction among hexoses and amino components leads to the formation of 5-hydroxymethylfurfural (5-HMF) (Shallenberger and Mattick, 1983) as intermediate. The HMF increase during processing and storage of fruit products was positively identified and quantified (Resnik and Chirife, 1987; Toribio and Lozano, 1979). Figure 7.8 shows the HMF increase of apple and grapefruit juice during prolonged storage. As Fig. 7.8 shows, the rate of accumulation can be divided into three periods. The first period is characterized as an induction time of approximately 2 weeks. During the second

7 . Fruit Products, Deterioration by Browning 175 Kcal/mol. 50 40 HMF (mg/100 g) 30 20 10 Apple, 37؇C Grapefruit, 40؇C 0 0 25 50 75 100 125 Time (days) Figure 7.8. Rate of accumulation of 5-HMF with time of storage in apple and grape juices (Saguy et al., 1978; Babsky et al., 1986). period the rate showed a rapid increase of HMF with a maximum at about 7–8 weeks. After that maximum the rate of formation diminished rapidly, and the HMF production approached a plateau. A similar behavior attributable to a second-order autocatalytic reac- tion (Frost and Pearson, 1961), was recognized by Schallenberger and Mattick (1983) during the acidic degradation of hexoses. It would appear that after 50 days of storage under the present conditions, HMF started to form brown pigments (melanoidins) in apple juice at such a rate that after some period the consumption equaled the formation via Amadori rearrangement of hexose degradation. Calculated activation energy/valid for the HMF formation in the range of temperatures considered, resulted Ea ¼ 35 Kcal=mol. Similar results were obtained by O’Beirne (1986) for apple juice concentrate. Petriella et al. (1985) found that the NEBr in different food systems reduced with decreasing pH. However, the range of work was pH ¼ 5 –7 and the authors showed an apparent change in the browning mechanism at pH ¼ 5 and lower. Although Wolfrom et al. (1974) also found that the browning rate decreased with pH, they worked with apple juice at pH ¼ 6 –7. It was speculated that an increase in the malic acid content was more effective in accelerating NEB than the consequent pH reduction. Concentration by evaporation ideally reduces costs and increases shelf life by removing water without changing the solid composition. However, in practice, clarified fruit juices are susceptible to color and flavor changes during evaporation. In the previous section we considered the reaction of the hexoses and amino components present in apple juice, leading to the formation of 5-hydroxymethylfurfural. HMF can also be produced by acid-catalyzed splitting of sugars (Shallenberger and Mattick, 1983). Extrapolation of the straight-line portion to the time axis, gives a value for the induction period. A similar induction period in the formation of HMF was observed by Shallenberger and Mattick (1983) and was attributed to some autocatalytic mechanism. Only an initial flat period and rapidly increasing rates at the outset of reaction were observed in apple juice (Toribio and Lozano, 1987). The formation of 5-HMF was also proposed to be used to complement color data in estimating the severity of heating during processing and storage of fruit juices (Askar,

176 Fruit Manufacturing EXAMPLE 7.1 NEB estimation during storage Urbicain and Lozano devised a nomogram for calculating the relative color increase from the initial one, with concentration (in Brix), time (in days), and temperature (in8C) given. This is shown in Fig. 7.9. In this example, the increase in color after 60 days’ of storage of a 708Brix apple juice concentrate at 308C needs to be estimated. A line is drawn passing through 708Brix and 60 days’ points, and extrapolated to the reference line. A second line is plotted joining the focus point (Fo) with the intersection between the first plotted line and the reference line. Finally, a line normal to temperature line, beginning at the intersection between (Fo-Ref) line and 308C line, indicates DC value at the corresponding scale. The result indicates an absorbance increase DC ¼ 0:68 can be estimated. 1984; Toribio and Lozano, 1987). The formation of HMF depends on the duration and temperature of processing and storage. In fresh, untreated juices the HMF content is prac- tically 0 (Babsky et al., 1986; Askar, 1984). The HMF level is important because it indicates the severity of heating that has been applied during processing, as reported for milk (Burton, 1984), honey (Jeuring and Kuppers, 1980), dehydrated apples (Resnik and Chirife, 1979), and tomato paste (Allen et al., 1980). Both HMF and furfural (2-furaldehyde) are useful as indicators of temperature abuse in orange juice (Meydav and Berk, 1978). 8Brix t days DC Ref T 8C Fo 65 5 Result: 100 10 DC = 067 20 25 30 1 37 200 15 Figure 7.9. Nomogram to estimate the relative color increase (DC) in Abs. 420 nm of concentrate as a function of time of storage or transportation, concentration (8Brix), and temperature.

7 . Fruit Products, Deterioration by Browning 177 EXAMPLE 7.2 NEB during refrigeration of a juice barrel 50-gallon plastic barrels, although practically displaced by high-volume containers, are still used in small processing plants. The problem is to estimate the necessary time to reduce the center temperature to values low enough to retard the NEB. Urbicain and Lozano (1992) found that by maintaining clarified concentrated apple juice (CAJ) for 60 min at 508C (outlet evaporator temperature), 10% increase in color and 5-HMF formation occurs. Barrels are filled at 508C to facilitate pumping and improve sanitary conditions. A typical barrel is 1 m high and has 0.55 m diameter. As viscosity increases during cooling from 0.06 Pas to 2.0 Pas, convective movement is rapidly restricted, and barrels are normally piled in threes. The well-known solutions for conductive cooling of an infinite cylinder can be applied (Carslaw and Jaeger, 1965; Welty et al., 1976). Assuming the following average properties for CAJ: r ¼ 1,360kg=m3 k ¼ 0:86 kcal=smC cp ¼ 0:64 kcal=kgC And considering a convective coefficient Hc as (Charm, 1963): Hc ¼ 0:095(Ti À Ta)rÀ1(BTU=h ftF) where r is the barrel radius. By solving analytically or graphically, temperature reduction at different barrel diameters was calculated and plotted in Fig. 7.10. Under normal storage condition, a CAJ barrel may need up to 2 days to reduce its temperature to sufficiently low NEB rate. It must be indicated that any efforts made to optimize the process will become useless if packaging and refrigeration is not properly done. The presence of excessive amounts of HMF is considered evidence of overheating. Askar (1984) indicated that HMF is also responsible for the cooked taste of apple juice. Multiple- effect evaporators were designed to concentrate apple juice at reduced temperatures, but in practice temperatures become very high in the initial effects (Lozano et al., 1984). Toribio and Lozano (1987) heated apple juice in a set of thin, rectangular cells, made of stainless steel, designed to have a relatively high sample capacity and a short come-up time (less than 40 s under the more adverse conditions) to evaluate the buildup of HMF in apple juices at the soluble solid concentrations and temperatures that usually prevail during concentration by evaporation. The various apple juices were heated in the range 100 –1088C for selected times (from 4 to 80 min). HMF, which has been shown to be essentially absent in fresh manufactured single- strength apple juice (Askar, 1984), increased to significant amounts during high-temperature heating. The relationship between buildup of HMF and time and temperature, and soluble solids is shown in Fig. 7.11. These results indicate that HMF increases linearly with time after an initial induction period, which depends on soluble solids and temperature. More research is needed to establish relationships between material properties and rates of oxidation, NEB, and enzymatic changes.

178 Fruit Manufacturing Temperature (؇C) 55 CAJ Barrel 45 distance from center r = 0.01 m r = 0.11 cm 35 r = 0.25 cm 25 15 5 −5 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 0 Time (h) Figure 7.10. Cooling rate of a 708Brix CAJ in barrel, stored in a –58C cooling chamber. The induction periods are comparable with the residence times in individual evaporatorHMF (mg/L) effects. Kurudis and Mauch (21) reported 32.5 min as the mean residence time in an industrial sugar evaporator similar to those commonly found in the fruit juice industry. 160 140 70؇Brix 108؇C 70؇Brix 100؇C 120 30؇Brix 108؇C 30؇Brix 100؇C 100 80 60 40 20 0 0 10 20 30 40 50 60 70 80 Time (min) Figure 7.11. HMF formation as a function of time and temperature, and soluble solids, for clarified apple juice (adapted from Toribio and Lozano, 1987).

7 . Fruit Products, Deterioration by Browning 179 The effect of variety on HMF formation was noticeable. The results show that the rate of buildup of HMF was especially dependent on juice composition. It was three to four times more rapid in Granny Smith than in Red Delicious apple juice. As noted previously, HMF can be formed either by heating of reducing sugars in acid solution, or by the reaction between hexoses and amino acids. Both pH and total amino acid content favor the formation of HMF in fruit juice. Calculated activation energies for HMF formation in apple juice and dehydrated apples ranged from 33.8 to 46.8 kcal/mol. (Resnik and Chirife, 1979; Toribio and Lozano, 1987). Toribio and Lozano (1987) considered 30 mg/l of HMF as a reasonable limit after heat treatment of apple juice. Time of heating required at various temperatures to attain this level is plotted in Fig. 7.12. The data in Fig. 7.12 indicate that variety has a more drastic effect on HMF buildup than on the development of NEB. Therefore, the measurement of HMF in fruit juice may provide a useful complement to color data. Depending on the composition of the juice, the HMF level can reach very high values. As HMF is an important intermediate in NEB via Maillard (Babsky et al., 1986), its production during processing may accelerate browning during storage. HMF content can then be used to complement color data in estimating the severity of heating during processing and the storage capacity of fruit juice concentrates. Nonenzymatic browning kinetics as affected by glass transition Although glass transition may control physical changes in foods, its effect on reaction kinetics is not well established. Significant issues are whether reactions become diffusion controlled and importance of glass transition on reaction rates. Lievonen et al. (1998) studied the effects of physical state, water plasticization, and glass transition on kinetics of NEB in a water solution, and concentrated polyvinylpyrrolidone (PVP) and maltodextrin (MD) model systems with 0.23, 0.33, and 0.44 aw at 248C with the same concentration of reactants, xylose, and lysine 1:1, (10%, w/w) in the water phase. Water contents of the MD and PVP systems increased from 6.3 to 9.7 and from 8.2 to 17.3 g H2O/ Time to attain 30 mg/L HMF (min) 100 Granny Smith Red Delicious 10 102 106 110 98 Temperature (؇C) Figure 7.12. Effect of temperature on rate of accumulation of 5-HMF in apple juice for Granny Smith (698Brix) and Red Delicious (70.68Brix) varieties (adapted from Toribio and Lozano, 1987).

180 Fruit Manufacturing 100 g dry matter, respectively, as aw increased from 0.23 to 0.44. The rate of NEB was the highest at all temperatures (10 –1008C) in water solution. The rate in PVP systems (Tg ranging from 30 to 608C) was higher than in MD systems (Tg ranging from 30 to 808C) both as a function of temperature at constant water content and as a function of water content at a constant temperature. Above Tg, reaction rates increased more rapidly than below Tg. These results may be useful in controlling NEB in processing and storage of concentrated food materials. REFERENCES Allen, B.H. and Chin, H.B. (1980). Rapid HPLC determination of hydroxymethylfurfural in tomato paste. J.Assoc. Off. Anal. Chem. 63: 1974 –1976. Askar, A. (1984). Flavor alterations during production and storage of fruit juices. Flussiges Obst. 11: 564 –569. Babsky, N., Toribio, J.L. and Lozano, J.E. (1986). Influence of storage on the composition of clarified apple juice concentrate. J.Food Sci. 51: 564 –567. Buedo, A. Elustondo, M.P. and Urbicain, M.J. (2001). Non enzymatic browning of peach juice concentrate. Innov. Food Sci. Emerg. Technol. 1: 255–260. Burton, H. (1984). Reviews of the progress of dairy science: the bacteriological, chemical, biochemical and physical changes that occur in milk at temperatures of 100 –1508C. Dairy Res. 51: 341–363. Carslaw, H.S. and Jaeger, J.C. (1969). Conduction of Heat in Solids, 2nd Edition. London: Oxford University Press, Inc. Charm, S. (1963). A Method For Experimentally Evaluating Heat-Transfer Coefficients In Freezers And Thermal Conductivity Of Frozen Foods. Food Technology 17: 1305. Cornwell, C.J. and Wrolstad, R.E. (1981). Causes of browning in pear juice concentrate during storage. J.Food Sci. 46: 515–518. Coseteng, M.Y. and Lee, C.Y. (1987). Changes in apple polyphenoloxidase and polyphenol concentrations in relation to degree of browning. J.Food Sci. 52: 985. Czapski. J. (1975). Wpliw wolnych aminokwasow na zmiany jal czageszczonych sokow iablkowYch podczas przechowy. Prze Mysi, Ferment I Rolny 8 –9: 19–23. del Castillo, M.D., Corzo, N, Polo, M.C., Pueyo, E. and Olano, A. (1998). Changes in amino acid composition of dehydrated orange juice during accelerated nonenzymic browning. J.Agric. Food Chem. 46: 277–280. Eichner, K. (1975). The influence of water content on non-enzymatic browning reactions in dehydrated foods and model systems. In Water Relations in Foods, Duckworth, R. (ed.). Academic Press, NY. Eichner, K. and Karel, M. (1972). The influence of water content on the amino browning reaction in model systems under various conditions. J.Agron. Food Chem. 20: 218–223. Fennema, O.R. (1985). Food Chemistry. New York, Mercel Decker, 991p. Frost, A.A. and Pearson, R.G. (1961). Kinetics and Mechanism, 2nd. ed. John Wiley & Sons, New York, NY. Hodge, J.E. (1953). Dehydrated foods. Chemistry of browning reactions in model systems. J.Agr. Food Chem. 1(15): 928–935. Jeuring, H.J. and Kuppers, F.J.E.M. (1980). High performance liquid chromatography of furfural and hydroxy- methylfurfural in spirits and honey. J.Assoc. Off. Anal. Chem. 63: 1215–1218. Johnson, G., Donnelle Y.J. and Johnson, D.K. (1969). Proantho-cyanidins as related to apple juice processing and storage. J.Food Sci. 33: 254 –257. Joslyn, M.A. and Ponting, J.D. (1951). Enzyme-catalyzed oxidative browning of fruit products. Adv. Food Res. 3: 1–7. Kacem, B., Cornell, J.A., Marshall, M.R., Shiremen, R.B. and Matthews, R.F. (1987). Nonenzymatic browning in aseptically packed orange drinks: effect of ascorbic acid, amino acids and oxygen. J.Food Sci. 52(6): 1668–1672. Kanner, J., Fishbein, J., Shalom, P., Harel, S. and Ben-Gera, I. (1982). Storage stability of orange juice concentrate packaged aseptic. J.Food Sci. 47: 429– 433. Koch, J. and Bretthauer, G. (1956). The vitamin C content of ripening fruits. Landwirstsch. Forsch. 9: 51–63. Labuza, T.P. and Riboh, D. (1982). Theory and application of Arrhenius kinetics to the prediction of nutrient losses in foods. Food Technol. 36(10): 66 –74. Labuza, T.P., Tannenbaum, S.R. and Karel, M. (1970). Water content and stability of low moisture and intermediate moisture of foods. Food Technol. 24: 543–550.

7 . Fruit Products, Deterioration by Browning 181 Lee, C.Y. and Jaworski, A. (1988). Phenolics and browning potential of white grapes grown in New York. Am. J. Enol. Vitic. 39: 337–340. Lee, C.Y., Kagan, V. Jaworski, A.W. and Brown, S.K. (1990). Enzymatic browning in relation to phenolic compounds and polyphenoloxidase activity among various peach cultivars. J.Agric. Food Chem. 38: 99–191. Lievonen, S.M., Laaksonen, T.J. and Roos, Y.H. (1998). Glass Transition and Reaction Rates: Nonenzymatic Browning in Glassy and Liquid Systems. J. Agric. Food Chem. 46(7): 2778–2784. Lozano, J.E. (1991). Kinetics of non enzymatic browning in model systems simulating clarified apple juice. Lebensm. Wiss. Technol. 24: 355–360. Lozano, J.E., Elustondo, M.P. and Romagnoli, J.A. (1984). Control studies in an industrial apple juice evaporator. J.Food Sci. 49: 1422–1427. Lozano, J.E., Biscarri R.D. and Ibarz, A. (1994). Enzymatic browning in apple pulps. J. Food Sci. 59: 1– 4. Marshall, M.R., Kim, J. and Cheng-I, W. (2000). Enzymatic Browning in Fruits, Vegetables and Seafoods. FAO. In http://www.fao.org/ag/ags /agsi/ENZYMEFINAL/. Meydav, S. and Berk Z. (1978). Colorimetric determination of browning precursors in orange juice products. J.Agric. Food Chem. 26: 282–285. O’Beirne, D. (1986). Effects of pH on non-enzymatic browning during storage in apple juice concentrate prepared from Bradley’s Seedling Apples. J.Food Sci. 51: 1073–1076. Ochoa, M.R. Kesseler, A.G., Vullioud, M.B. and Lozano J.E. (1999). Physical and chemical characteristics of raspberry pulp: storage effect on composition and color. Lebensm. Wiss. Technol. 32(3): 149–153. Petriella, C., Resnik, S.L., Lozano, R.D. and Chirife, J. (1985). Kinetics of deteriorative reactions in model food systems of high water activity: color changes due to non-enzymatic browning. J.Food Sci. 50: 625–630. Ponting, J.D and Joslyn, M.A. (1948). Ascorbic acid oxidation and browning in apple tissue extracts. Arch. Biochem. 19: 47–51. Pribella, A. and Betusowa, M. (1978). Veranderungen in Geha Sticktoffhaltingen Soffen bei der Lagerung von Obstsaft-ko traten. Fruchtsaft-lndustrie 9(1): 15–19. Resnik, S. and Chirife, J. (1979). Effect of moisture content and temperature on some aspects of non-enzymatic browning in dehydrated apple. J.Food Sci. 44: 601–606. Reynolds, T.H. (1965). Chemistry of non-enzymatic browning II. Adv. Food Res. 14: 167–210. Saguy, L., Kopelman, I.J. and Mizrahi, S. (1978). Extent of nonenzymatic browning in grapefruit juice during thermal and concentration processes: Kinetics and prediction. J. Food Proc. Pres 175–184. Sapers, G.M. and Douglas Jr., F.W. (1987). Measurement of enzymatic browning at cut surfaces and in juice of raw apple and pear fruits. J. Food Sci. 52: 1258–1263. Shallenberger, R.S. and Mattick, L.R. (1983). Relative stability of glucose and fructose at different acid pH. Food Chem. 12: 159–166. Shannon, C.T. and Pratt, D.E. (1967). Apple polyphenol oxidase activity in relation to various phenolic compounds. J. Food Sci. 32: 479–483. Spanos, G.A. and Wrolstad, R.E. (1992). Phenolics of apple, pear, and white grape juice and their changes with processing and storage: a review. J.Agric. Food Chem. 40: 1478–1487. Spark, A.A. (1969). Role of amino acids in nonenzymatic browning. J.Sci. Food. Agric. 20(5): 308–314. Toribio, J.L. and Lozano, J.E. (1984). Nonenzymatic browning in apple juice concentrate during storage. J.Food Sci. 49: 889–892. Toribio, J.L. and Lozano, J.E. (1986). Heat induced browning of clarified apple juice at high temperatures. J.Food Sci. 51: 172. Toribio, J.L. and Lozano, J.E. (1987). Formation of 5-hydroxymethylfurfural in clarified apple juice during heating at elevated temperatures. Lebensm. Wiss. Technol. 20: 59–63. Toribio, J.L., Nunes, R.V. and Lozano, J.E. (1984). Influence of water activity on the nonenzymatic browning of apple juice concentrate during storage. J.Food Sci. 49: 1630 –1632. Urbicain M.J., and Lozano, J.E. (1992). Damage of concentrated apple juice during processing and storage. Lebensm. Technol. (25), 194–204. Va´mos-Vigya´zo´ , L. (1981). Polyphenol oxidase in fruits and vegetables. CRC Crit. Rev. Food Sci. Nutr. 15: 49–127. Waletzko, P. and Labuza, T.P. (1976). Accelerated shelf-life testing an intermediate moisture food in air and in an oxygen free-free sphere. J. Food Sci. 41: 1338. Walker, J.R.L. (1995). Enzymatic browning in fruits: Its biochemistry and control. In Enzymatic Browning and Its Prevention, Lee, C.Y. and Whitaker, J.R. (eds.). ASC Symposium Series 600, American Chemical Society, Washington, DC, pp. 8–22. Wang, T., Gonzalez, A.R., Gbur, E.E. and Aselage, J.M. (1993). Organic acid changes during ripening of processing peaches. J. Food Sci. 58: 631–632.

182 Fruit Manufacturing Warmbier, H.C., Schnickels, R.A. and Labuza, T.P. (1976). Non-enzymatic browning kinetics in an intermediate moisture model system. Effect of glucose to lysine ratio. J.Food Sci. 41: 981–983. Welty, J.R., Wicks, C.E. and Wilson, R.E. (1976). Fundamentals of Momentum, Heat and Mass Transfer, 2nd ed., Wiley. Whitaker, J.R. and Lee, C.Y. (1995). Recent advances in enzymatic browning. In Enzymatic Browning and Its Prevention, Lee, C.Y. and Whitaker, J.R. (eds.). American Chemical Society Symposium Series 600, 2–7. American Chemical Society, Washington, DC. Wolfrom, M.L., Schuertz, R.D. and Cavalieri, L.F. (1974). Factors affecting the Maillard browning reaction between sugars and aminoacids. Studies on the non-enzymatic browning of dehydrated orange juice. J.Agr. Food Chem. 22: 796 –801.

CHAPTER 8 INHIBITION AND CONTROL OF BROWNING 8.1. INTRODUCTION As previously discussed in this book (Chapter 6) fruits are complex systems, which, after size reduction (pulping, milling, or cutting) are transformed into a mixture of chemical and biochemical active components reacting in aqueous media. Moreover, process conditions cover a wide range of temperatures (Fig. 8.1), which make the modeling and prediction of deteriorative reactions even more difficult. Voluminous literature on the interplay of these parameters during processing of foodstuffs is available. Browning of fruits is a major problem in the fruit industry and is believed to be one of the main causes of quality loss during postharvest handling and processing. The mechanism of browning in fruits and fruit products is well characterized and can be enzymatic or none- nzymatic in origin (Chapter 7). 8.2. INHIBITION AND CONTROL OF ENZYMATIC BROWNING Enzymatic browning (EB) is the result of fast reactions. Even an optimized processing technology cannot completely avoid the EB during pulping and pressing of fruit juice, unless special care is taken to avoid oxygen. Enzymes and reactions responsible of discoloration in fruits are described in Chapter 7. In brief, during EB reactions, polyphenol oxidase catalyzes the oxidation of phenols to o-quinones, which are highly reactive compounds. o-Quinones thus formed undergo spontaneous polymerization to produce high molecular weight com- pounds or brown pigments (melanins). These melanins may in turn react with amino acids and proteins leading to enhancement of the brown color produced. Many studies have focused on either inhibiting or preventing polyphenol oxidase activity in foods. Va´mos-Vigya´zo´ (1995) classified the principles of EB prevention into: . Inhibition or inactivation of the enzyme, and . Elimination or transformation of the substrate. The author indicated that it is not easy to classify an inhibitor as belonging only to one of these categories. Moreover, many inhibitors act on both enzyme and substrate. It must be emphasized that EB in fruits and fruit products can be controlled or reduced also by: . Selecting cultivates of slight browning tendency. . Improving the agricultural techniques. . Identifying PPO activity, phenolic composition, and browning kinetics. 183

184 Fruit Manufacturing Freeze drying & freeze −358C Concentration Removal of heat Freezing Chilling Storage Ambient Raw material preparation temperature Size reduction Mechanical separation Enzyme treatment Use of membranes Application Dehydration of Blanching Pasteurization heat Evaporation Ultra high temperature Processes 150 8C Figure 8.1. Range of temperatures during typical fruit processing and storage. Various techniques and mechanisms have been developed for controlling EB. These techniques attempt to eliminate one or more of the essential components (oxygen, enzyme, copper, or substrate) from the reaction. As EB is an oxidative reaction, it can be retarded by the elimination of oxygen from the cut surface of the fruit. However, browning restarts rapidly when oxygen is reintroduced. Oxygen exclusion is possible by immersion in syrup, deoxygenated water, or the coating of fruit with films not permeable to that gas (McEvily et al., 1992). As copper prosthetic group of polyphenol oxidases must be present for the EB reaction to occur, these chelating agents capable of removing Cu may be effective to control ED deterioration. Inactivation of the polyphenol oxidase by heat treatments, such as steam blanching, is effectively applied for the control of browning in fruits that are to be canned or frozen. Chemical modification of phenolic substrates, such as chlorogenic acid, caffeic acid, and tyrosine, can however prevent oxidation. Certain chemical compounds react with the products of polyphenol oxidase activity and inhibit the formation of the colored com- pounds produced in advanced, nonenzymatic reaction steps, which finally lead to the forma- tion of brown compounds. Other techniques, such as the use of naturally occurring enzyme inhibitors and ionizing radiation, have been used as alternatives to heat treatment and the health risks associated with certain chemical treatments. It must be realized that inactivation of enzymes responsible for browning in fruits can be irreversible (e.g., heat treatment) or reversible (e.g., use of ascorbic acid). A general classification of the methods used to inhibit EB is sketched in Fig. 8.2. 8.2.1. Thermal Treatments 8.2.1.1. Elevated Temperatures Although steam blanching is one of the most effective methods for controlling EB in canned or frozen fruits (Va´mos-Vigya´zo´ , 1995), it is not a practical alternative for treatment of fresh

8 . Inhibition and Control of Browning 185 Thermal inactivation EB Inhibition and control Chemical Nontraditonal treatment methods Figure 8.2. General classification of methods for inhibition of enzymatic browning. foods. In such cases, the exclusion of oxygen and/or the application of inhibitors should be considered. Moreover, blanching should not be used as it affects the texture and flavor of fruit products. Adams (1991) reviewed enzyme inactivation during heat processing of foodstuffs. He concluded that enzymes have complex covalent and noncovalent structures, which are susceptible to heat-induced chemical degradation and disruption. In general enzyme inacti- vation as a function of temperature can be described by the Arrhenius or activated-complex model. It is also known that refrigeration (0– 48C) retards browning; however during fruit juice processing the cellular tissue is practically destroyed, and low temperatures are not enough to control oxidation. It is also true that about 10 s at 908C inactivates PPO (Dimick et al., 1951), which are conditions easily provided during heating of pulps. However, in practice a long delay occurs between crushing (or pulping) and thermal processing. Yemeniciog¨ılu et al. (1997) studied the heat-inactivation kinetics of crude polyphenol oxidase (PPO) from six apple cultivars (Golden Delicious, Starking Delicious, Granny Smith, Gloster, Starckinson, and Amasya) at three temperatures (688, 738, and 788C). PPO activity initially increased and then decreased with heat, following a first-order kinetic model (Fig. 8.3). The authors attributed the increase in activity to the presence of latent PPO. Calculation of activation energies (54.7–77.2 kcal/mol) indicated that PPO in apples was generally more heat stable than PPO in other fruits, like banana (Galeazzi et al., 1981), grape (Lee et al., 1983), and pear (Halim and Montgomery, 1978). Thermal enzymatic inactivation is described in accordance with kinetic parameters such as decimal reduction times (D), inactivation rate constant (k), z values (z), and activation energies (Ea). The D value, or decimal reduction value, is defined as the time required to inactivate 90% of the original enzyme activity at a given temperature. An inactivation reaction, which follows first-order kinetics, has a D value equivalent to 2.303/k. Temperature dependence of the D value is given by the z value, which represents the temperature increase required in order to obtain a 10-fold (1-log cycle) decrease in D value. For a first-order decay process, the D value is equivalent to ln (10) k. Similar to the z value is the activation energy (Ea), which expresses the temperature dependence of the k value as indicated in the Arrhenius relationship: ln k ¼ ÀEa=RT þ ln A k ¼ A(eÀEa=RT )

186Activity, as % of original activity Fruit Manufacturing 1000 68؇C 73؇C 100 78؇C 10 10 20 30 40 50 0 Time (min) Figure 8.3. Effect of heating time and temperature on PPO in apple (Gloster cultivar) (Yemeniciog¨ılu et al., 1997) with permission. The Q10 value is the change in the rate of a reaction that occurs with a 108C change in temperature and can be related to the Arrhenius equation as, Q10 ¼ e10Ea=RT (Tþ10) (Labuza and Riboh, 1982). While chemical reactions of single polyphenols have been described step by step, in complex food systems only secondary effects, such as color development, can be recorded. Color parameter was useful for studying the kinetics of EB reactions. Sapers and Douglas (1987) reported that decreases in the CIE LÃ value correlated well with increases in fruit browning. Labuza et al. (1990) proposed the normalized DL=L0(%) values as a measure of browning, when initial Hunter L0 values varied slightly between samples. Genovese et al. (1997) used deviation from initial Hunter parameters to study the EB in cloudy apple juice. These authors prepared different types of samples: natural juice (without steam treatment during crushing), not centrifuged (NJnC); natural juice, centrifuged (NJnC); cloudy juice (steam treated), not centrifuged (CJnC); and cloudy juice, centrifuged (CJnC). Figure 8.4 shows the variation of DL ¼ L À L0 with time, for the different cloudy apple juice assayed. Luminosity decreases monotonically in the case of centrifuged natural juice (NJC) and remains practically constant for cloudy juices, either centrifuged (CJC) or not (CJnC). The small increase in DL for CJnC samples was attributed to partial precipitation of insoluble particles. Not centrifuged natural juices (NJnC) showed a completely different behavior and the rate of luminosity variation could be divided into two periods. The first period was characterized as a rapid increase in DL, attributed to the fast precipitation of unstable particles. During the second period, after a maximum at 10 min the luminosity decreased exponentially due to the oxidative darkening. The combined effect of particle precipitation and EB resulted in a strong nonlinear behavior. Hunter hue angle and saturation index (Chapter 5) was practically constant in all cases other than natural juices without treatments, in this case it is attributable to particle

8 . Inhibition and Control of Browning 187 8 NJnC NJC NJnC CJnC CJC 6 Precipitation 4 Enzymatic DL browning 2 CJnC 0 CJC NJC 2 0 10 20 30 40 50 60 Time (min) Figure 8.4. Variation of DL with time, for the different samples assayed (Genovese et al., 1997) with permission. precipitation. While hue angle decreases, the saturation index increases with time for NJnC samples. The color difference (DE) development in apple juice samples is shown in Fig. 8.5. Analysis of the data concerning the color deterioration of apple juice suggests that cloud characteristics and EB effect on hue should not be independently considered. Moreover, steam treatment of juice was very effective not only in inactivating oxidative enzymes, but also in stabilizing cloudiness. Similar results were obtained by McKenzie and Beveridge (1988), during the blanching of Spartan apple juice. The authors attributed apple particulate stabilization to the formation of a protective colloid that prevented aggregation. It was also observed that centrifugation (4,200g per 5 min) had a positive effect in controlling, or at least in retarding, color changes, when applied to natural juices without heat treatment (NJC). Table 8.1 lists some thermal fruit treatments for the inhibition of EB.

188 Fruit Manufacturing 12 NJnC NJC 10 CJnC CJC 8 DE 6 4 2 0 0 10 20 30 40 50 60 Time (min) Figure 8.5. Total color difference (DE) as a function of time, for natural and cloudy apple juices, at 208C (Genovese et al., 1997) with permission. 8.2.1.2. Refrigeration Temperatures In general, for every 108C reduction in temperature a similar decrease in the rate of enzyme- catalyzed reactions occurs, which is referred to as the temperature coefficient (Q10). This effect was attributable to a decrease in both mobility and ‘‘effective collisions’’ necessary for the formation of enzyme–substrate complexes and their products. Freezing temperatures of À188C or below are often used for the long-term preservation of food. Some fruits (berries) may be precooled or stored at chilling temperatures. However, others like bananas, mangoes, and avocados are susceptible to chill injury and should Table 8.1. Inhibition of EB by thermal treatment. Fruit/product Inhibition method Reference Apricot substrate is catechin 808C at 10 min Dijkstra and Walker (1991) and chlorogenic acid 658C at 20 min Siddiq et al. (1994) Plum juice Steam heating of the mash Genovese et al. (1997) Cloudy apple juice in the range 65 –70C for 15–20 s

8 . Inhibition and Control of Browning 189 therefore not be stored below their respective critical temperatures (Fennema, 1975). Cold preservation and storage during distribution and retailing are necessary for the prevention of browning in fruit, since refrigerated temperatures are effective in lowering polyphenol oxidase activity. 8.2.2. Chemical Inhibition Chemical antibrowning agents have been commonly used to prevent browning of fruits and fruit products. Antibrowning agents are compounds that either act primarily on the enzyme or react with the substrates and/or products of enzymatic catalysis in a manner that inhibits colored product formation. The enzyme PPO can be inhibited by acids, halides, phenolic acids, chelating agents, sulfites, reducing agents such as ascorbic acid, quinone couplers such as cysteine, and some other substrate-binding compounds. Figure 8.6 shows the evolution of the chemical methods for the inhibition of EB applied to fruit products. The most widespread methodology used in the fruit industry for control of EB is the addition of sulfiting agents. The major effect of sulfites on EB is described in Fig. 8.7. As a reducing agent, sulfites reduce the o-quinone produced by PPO catalysis to the less reactive diphenol, preventing the development of later condensation of complex brown melanins. Inactivation of PPO by application of sulfur dioxide (SO2) has been successful in preventing EB, but its use was restricted by regulations. Sulfites have been linked to allergic reactions, the Food and Drug Administration (FDA) prohibited the use of sulfite preservatives in fresh vegetables and fruits (Langdon, 1987). The effect of reducing agents is temporary because these compounds oxidize irreversibly by reaction with pigments, enzymes, and metals. Their role is based in their ability to reduce o-quinones (Fig. 8.7). Sulfydryl compounds transform o-quinones in stable, colorless products. More More traditional innovative Cyclodextrin EDTA Chitosan Organic acids Chelating agents Sulfiting Reducing Chemical Aromatic Hexylresorcinol Cysteine agents inhibition enzyme Aliphatic alcohols Glutathione inhibitor Ascorbic of EB Anions acid and analogs Proteolitic Ficin enzymes Papain Bromelain Acidulant Peptides Carbohydrate Honey derivatives Citric acid Phosphoric acid Figure 8.6. Description of chemical methods for the inhibition of EB.

190 Fruit Manufacturing Reducing agent OH OH O PPO + O2 PPO + O2 Melanins R R OH RO Monophenol Diphenol o-Quinone Figure 8.7. Effect of reducing agents on the first stages of EB. Alternative inhibitors of PPO were investigated extensively (Shannon and Pratt, 1967; Park and Luh, 1985; Sapers and Ziolkowski, 1987; Oszmianski and Lee, 1990; Siddiq et al., 1994; Lozano-de-Gonzalez et al., 1993). Tables 8.2–8.4 list some alternatives to sulfite antibrowning agents, according to their primary mode of action (McEvily et al., 1992): reducing agents, chelating agents, enzyme inhibitors, complexing agents, and miscellaneous methods. Different EB inhibitors were assayed in raw apple juice and on cut surfaces of apple plugs, using quantitative measurements of color changes to evaluate treatment effective- ness during storage by Sapers et al. (1989). While ascorbic acid-6-fatty acid esters showed Table 8.2. Nonsulfite antibrowning agents applied to fruits and fruit products. Reducing agents. Name Mechanism of inhibition Comments Reference Ascorbic acid Free radical scavenger. Effect on PPO activity is controversial. Gola-Goldhirsh Reduces o-quinone It is easily decomposed to form et al. (1984) to diphenols. dehydroascorbic acid. Insufficient penetration into the cellular Janovitz-Klapp Erythorbic acid Idem matrix of fruits et al. (1990) Ascorbyl phosphate Releases ascorbic acid Erythorbic and ascorbic acid Borestein (1965) esters (APE) when hydrolyzed by application depends on the fruit. and ascorbyl fatty acid phosphatases One compound cannot be Seib and Liao acid esters (AFAE) substituted for the other (1987) without prior evaluation Sulfydryl compounds These agents react Sapers et al. (1989) with o-quinones In APE inhibition power depends Sapers et al. (1991) to produce stable, on the acidity of the fruit and Pierpoint (1966) colorless products the activity of endogenous acid phosphatase. AFAE needs emulsifying agents, which have detrimental effect on antibrowning ability This category is reduced to sulfur-containing amino acids (e.g., cysteine and methionine). High concentrations affect taste of treated fruit products

8 . Inhibition and Control of Browning 191 Table 8.3. Nonsulfite antibrowning agents applied to fruits and fruit products: Chelating agents and enzyme inhibitors. Name Mechanism of inhibition Comments Reference Ethylenediamine Chelating agents bind EDTA or its sodium salt is used McEvily et al. (1992) tetraacetic acid to the active site of in the food industry as PPO, or reduce Cu a metal chelating agent Phosphate-based agents availability for the enzyme Acidic polyphosphate mixture Ashoor and Zent has been evaluated as EB (1984) Idem inhibitor in combination with ascorbic acid Frankos et al. (1991) Substituted resorcinols 4-hexylresorcinol inhibits browning, is water soluble, stable, and nontoxic (GRASS) Table 8.4. Nonsulfite antibrowning agents applied to fruits and fruit products. Miscellaneous agents. Name Mechanism of inhibition Comments Reference Enzyme o-Methyl transferase converts Method too expensive Finkle and Nelson (1963) treatments PPO substrates to ferulic acid Martinez et al. (1986) (inhibitor of PPO) The order of decreasing Anions inhibition power Janovitz-Klapp et al. (1990) Sodium, calcium, and zinc of halides is F > Cl > Br > I chloride are pH-dependent inhibitors of PPO, explained by the interaction between charges of halides and active site of PPO antibrowning activity in juice, ascorbic acid-2-phosphate (AAP) and -triphosphate was effective for cut fruit surfaces. Combinations of ascorbic acid (AA) with an acidic polyphosphate were highly effective with both juice and cut surfaces. Cinnamate and benzoate inhibited browning in juice, but induced browning when applied to cut surfaces. On the contrary, combinations of beta- cyclodextrin with AA were effective in juice, but not on cut surfaces. Sapers (1991) infiltrated ascorbic acid-2-phosphate (AAP) and ascorbic acid into apple tissue to control browning. AAP hydrolysis by endogenous acid phosphatase (APase) yielded AA, which became oxidized to dehydroascorbic acid. APase activity varied greatly with commodity, method of sample preparation, and sample pH. Variation in the ability of AAP to inhibit browning in different products could be explained by these factors. Montgomery (1983) treated pear juice concentrates with 0.2 mM cysteine and observed color changes of concentrates during storage for 6 months at different temperatures. Initial browning of concentrates was eliminated by cysteine treatment of pear juice. Cysteine appeared to retard Maillard reaction in pear juice concentrates and no deleterious changes in flavor intensity were noted. During milling and finishing operations EB is difficult to control even with high levels of SO2 or vitamin C because of the incorporation of air. Regarding opalescent and cloudy juices,